Substituted cyclopentane compounds useful as neuraminidase inhibitors

ABSTRACT

Compounds represented by formula (I), and pharmaceutically acceptacle salts thereof; and their method of preparation are provided. Compounds of the above formula are influenza virus neuraminidase inhibitors and can be used in treating patients infected with influenza virus.

DESCRIPTION

This application claim benefit to provisional application 60/019,930Jun. 14, 1996 and this application claim benefit to provisionalapplication 60/044,010, May 2, 1997.

TECHNICAL FIELD

This invention relates to novel substituted cyclopentane compounds andderivatives thereof useful as neuraminidase inhibitors, topharmaceutical compositions containing said compounds useful for theprevention, treatment or amelioration of viral, bacterial and otherinfections, and to methods of using said compounds. The presentinvention is also concerned with novel intermediates or precursors forproducing the novel substituted cyclopentane compounds of the presentinvention.

BACKGROUND OF THE INVENTION

Despite the wealth of information available, influenza remains apotentially devastating disease of man, lower mammals, and birds. Noeffective vaccine exists and no cure is available once the infection hasbeen initiated.

Influenza viruses consist of eight pieces of single stranded RNA,packaged in orderly fashion within the virion. Each piece codes for oneof the major viral proteins. The replication complex is enclosed with amembrane composed of matrix protein associated with a lipid bilayer.Embedded in the lipid bilayer are two surface glycoprotein spikes,hemagglutinin (HA) and the enzyme neuraminidase (NA). All of the viralgenes have been cloned and the three-dimensional structures of thesurface glycoproteins have been determined.

Influenza viruses continually undergo antigenic variation in the twosurface antigens, HA and NA, toward which neutralizing antibodies aredirected. For this reason, vaccines and a subject's natural immunesystem have not been very effective. Attention is now being directed tofinding other potential antiviral agents act ing at other sites of thevirion. This invention is directed to novel compounds which are usefulin inhibiting the viral surface enzyme NA.

Furthermore, many other organisms carry NA. Many of these NA-possessingorganisms are also major pathogens of man and/or mammals, includingVibraeo cholerae, Clostridium perfringes, Streptococcus pneumonia,Arthrobacter sialophilas, and other viruses, such as parainfluenzavirus, mumps virus, Newcastle disease virus, fowl plague virus, andSendai virus. Compounds of this invention are also directed toinhibiting NA of these organisms.

In viruses, NA exists as a tetramer made of four roughly sphericalsubunits and a centrally-attached stalk containing a hydrophobic regionby which it is embedded in the organism's membrane. Several roles havebeen suggested for NA. The enzyme catalyzes cleavage of the α-ketosidiclinkage between terminal sialic acid and an adjacent sugar residue.Removal of the sialic acid lowers the viscosity and permits access ofthe virus to the epithelial cells. NA also destroys the HA receptor onthe host cell, thus allowing elution of progeny virus particles frominfected cells.

Research indicates that the active site for influenza neuraminidaseremains substantially unchanged for the major strains of influenza. Forexample, a comparison of sequences from influenza A subtypes andinfluenza B shows conserved residues with crucial structural andfunctional roles. Even though the sequence homology is only about 30%,many of the catalytic residues are conserved. Furthermore, thethree-dimensional structures of influenza A and B neuraminidases havebeen determined. Superposition of the various structures showsremarkable structural similarity of the active site. Since the activesite amino acid residues are conserved in all known influenza Aneuraminidases that have been sequenced so far, an inhibitor that iseffective against different strains of influenza A and/or Bneuraminidase can be designed based on the three-dimensional structureof a neuraminidase.

In general, the role of NA is thought to be for the mobility of thevirus both to and from the site of infections. Compounds that inhibitneuraminidase's activity may protect a subject from infection and/orcure a subject once infection has set in. It is a further object of thisinvention to provide a method of using compounds of this invention fortreating and/or curing a viral infection.

Analogs of neuraminic acid, such as2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) and its derivativesare known to inhibit HA in vitro; however, these compounds are inactivein vivo. Palese and Schulman, in CHEMOPROPHYLAXIS AND VIRUS INFECTION OFTHE UPPER RESPIRATORY TRACT, Vol. 1 (J. S. Oxford, Ed.), CRC Press,1977, at PS 189-205.

Von Itzstein et al. (PCT Publication WO 91/16320) describes cyclohexaneanalogs of α-D-neuraminic acid of the formula

wherein:

A is O, C or S in Formula (a), and N or C in Formula (b);

R¹ is CO₂H, PO₃H₂, NO₂, SO₂H, SO₃H, tetrazolyl-, CH₂CHO, CHO, orCH(CHO)₂;

R² is H, OR⁶, F, Cl, Br, CN, NHR⁶, SR⁶ or CH₂X, where X is NHR⁶ halogen,or OR⁶;

R³ and R³′ are H, CN, NHR⁶, SR⁶, ═NOR⁶, OR⁶, guanidino, NR⁶;

R⁴ is NHR⁶, SR⁶, OR⁶, CO₂R⁶, NO₂, C(R⁶)₃, CH₂CO₂R⁶, CH₂NO₂ or CH₂NHR⁶;

R⁵ is CH₂YR⁶, CHYR⁶CH₂YR⁶ or CHYR⁶CHYR⁶CH₂YR⁶;

R⁶ is H, acyl, alkyl, allyl, or aryl;

Y is O, S, NH, or H;

and parmaceutical salts thereof, useful as antiviral agents

In addition, certain benzene derivatives are suggested in U.S. Pat. No.5,453,533 as being inhibitors of influenza virus neuraminidase andvarious others are disclosed in U.S. patent application Ser. No.08/413,886. Yamamoto et al. describe various sialic acid isomers ashaving inhibitory activity against neuraminidase in Synthesis of SialicAcid Isomers With Inhibitory Activity Against Neuraminidase, TETRAHEDRONLETTERS, Vol. 33, No. 39, pp. 5791-5794, 1992.

WO 96/26933 to Gilead Sciences, Inc. describes certain 6-membered ringcompounds as possible inhibitors of neuraminidase.

However, none of these references disclose the cyclopentane derivativesof the present invention.

SUMMARY OF INVENTION

An aspect of the present invention is directed to compounds representedby the formula:

wherein

X is CH₂, O or S;

R₁ is H, OH, NH₂, or OR₁₁;

R₉ is CO₂H, SO₃H, PO₃H₂, NO₂, esters thereof, or salts thereof;

R₂ is H,

each of R₃ and R₈ individually is H, (CH₂)_(n)CO₂R₁₀, (CH₂)_(m)OR₁₀,CON(R₁₀)_(m), (CH₂)_(n)N(R₁₀)_(m), CH(R₁₀)_(m), (CH₂)_(n)(R₁₀)_(m),CH₂CH(OR₁₀)CH₂OR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂OR₁₀, CH₂OR₁₀, CH(OR₁₀)CH₂NHR₁₀,CH₂CH(OR₁₀)CH₂NHR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂NHR₁₀, orNR₁₀C(═NR₁₀)N(R₁₀)_(m); provided that at least one of R₂, R₃ and R₈ isother than H;

R₄ is H, (CH₂)_(n)OH, (CH₂)_(n)NH₂, (CH₂)_(n)C(═NH)NH₂,(CH₂)_(n)NHC(═NR₇)NH₂, (CH₂)_(n)CN or (CH₂)_(n)N₃;

R₅ is H, lower alkyl, branched chain alkyl, cyclic alkyl or CF₃;

R₇ is H, OH, CN, NH₂ or NO₂;

each R₁₀ individually is H, lower alkyl, lower alkylene, branched alkyl,cyclic alkyl, substituted cyclic alkyl, (CH₂)_(n) aromatic,(CH₂)_(n)-substituted aromatic, and when m is 2 both R₁₀ groups can alsobe interconnected to form an N-heterocyclic ring;

R₁₁ is lower alkyl, branched alkyl, or (CH₂)_(m) aromatic;

m is 1 or 2; and

n is 0-4; and

further provided that when X is O or S, R₃ and R₈ is other thanCH(OR₁₀)CH(OR₁₀)CH₂OR₁₀;

and phramaceutically acceptable salts thereof.

The present invention is also concerned with compositions for inhibitinginfluenza virus neuraminidase comprising a pharmaceutically acceptablecarrier and an amount effective for inhibiting influenza virusneuraminidase of a compound as defined above.

A further aspect of the present invention involves a method forinhibiting influenza virus that comprises administering to a patient inneed thereof a compound as defined above in an amount effective forinhibiting influenza virus neuraminidase.

A still further aspect of the present invention is concerned withtreating influenza virus infection comprising administering to a patientin need thereof a compound as defined above in an amount effective forinhibiting influenza virus neuraminidase.

The present invention is also concerned with methods for producing thecompounds defined above.

Best and Various Modes for Carrying Out Invention

An aspect of the present invention is directed to compounds representedby the formula:

wherein

X is CH₂, O or S;

R₁ is H, OH, NH₂, or OR₁₁;

R₉ is CO₂H, SO₃H, PO₃H₂, NO₂, esters thereof, or salts thereof;

R₂ is H,

each of R₃ and R₈ individually is H, (CH₂)_(n)CO₂R₁₀, (CH₂)_(m)OR₁₀,CON(R₁₀)_(m), (CH₂)_(n)N(R₁₀)_(m), CH(R₁₀)_(m), CH₂)_(n)(R₁₀)_(m),CH₂CH(OR₁₀)CH₂OR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂OR₁₀, CH₂OR₁₀, CH(OR₁₀)CH₂NHR₁₀,CH₂CH(OR₁₀)CH₂NHR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂NHR₁₀, orNR₁₀C(═NR₁₀)N(R₁₀)_(m);

provided that at least one of R₂, R₃ and R₈ is other than H;

R₄ is H, (CH₂)_(n)OH, (CH₂)_(n)NH₂, (CH₂)_(n)C(═NH)NH₂,(CH₂)_(n)NHC(═NR₇)NH₂, (CH₂)_(n)CN or (CH₂)_(n)N₃;

R₅ is H, lower alkyl, branched chain alkyl, cyclic alkyl or CF₃;

R₇ is H, OH, CN, NH₂ or NO₂;

each R₁₀ individually is H, lower alkyl, lower alkylene, (CH₂)_(n)aromatic, branched alkyl, cyclic alkyl, substituted cyclic alkyl,(CH₂)_(n)-substituted aromatic, and when m is 2 both R₁₀ groups can alsobe interconnected to form an N-heterocyclic ring;

R₁₁ is lower alkyl, branched alkyl, or (CH₂)_(m) aromatic;

m is 1 or 2; and

n is 0-4; and

further provided that when X is O or S, R₃ and R₈ is other thanCH(OR₁₀)CH(OR₁₀)CH₂OR₁₀;

and pharmaceutically acceptable salts thereof.

Concerning R₁₀ when m=2, each R₁₀ can be the same or different.

The lower alkyl groups contain 1 to about 8 carbon, and preferably 1 toabout 3 carbon atoms, and can be straight, branched-chain or cyclicsaturated aliphatic hydrocarbon groups.

Examples of suitable alkyl groups include methyl, ethyl and propyl.Examples of branched alkyl groups include isopropyl and t-butyl.Examples of suitable cyclic aliphatic groups typically contain 3-8carbon atoms and include cyclopentyl and cyclohexyl. The aromatic oraryl groups are preferably phenyl or alkyl substituted aromatic groups(aralkyl) such as phenyl C₁₋₃ alkyl such as benzyl.

Examples of substituted cycloalkyl groups include cyclic aliphaticgroups typically containing 3-8 carbon atoms in the ring substitutedwith alkyl groups typically having 1-6 carbon atoms and/or hydroxygroup. Usually 1 or 2 substituted groups are present.

The lower alkylene group can be straight, branched chain or cyclicunsaturated hydrocarbon group and contains 2-8 carbon atoms andpreferably 2-3 carbon atoms. Examples of alkylene groups are vinyl,1-propenyl, allyl, isopropenyl, 2-methyl-2-propenyl and cyclopentenyl.

The N-heterocyclic rings contain 3-7 atoms in the ring. The heterocyclicrings can be substituted such as with a lower alkyl group. Examples ofsuitable heterocyclic groups are pyrrolidino, azetidino, piperidino,3,4-didehydropiperidino, 2-methylpiperidino and 2-ethylpiperidino.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutially acceptable, inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicyclic, succinic,toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic,benzoic, malonic, naphthalene-2-sulphonic, trifluoroacetic andbenzenesulphonic acids.

Salts derived from appropriate bases include alkali such as sodium andammonia.

Examples of some specific compounds within the scope of the presentinvention are:

cis-3-[(methylcarbonylamino)methyl]cyclopentanecarboxylic acid;

trans-3-amino-c-⁴-(methylcarbonylamino)methyl-r-cyclopentanecarboxylicacid;

trans-3-{[(amino)(imino)methyl]amino}-c-4-[(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid;

4(3-{[(amino)(imino)methyl]amino}-3α-[(2-hydroxy-1-methylcarbonyl-amino)ethyl]-1-cyclopentanecarboxylicacid;

sodium3β-{[amino)(imino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylate;

trans-3-amino-trans-1-hydroxy-cis-4[(hydroxymethyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid;

trans-3-{[(amino)(imino)methyl]amino}-trans-1-hydroxy-cis-4-[(2-hydroxymethyl)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylicacid;

3β-amino-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentancarboxylicacid;

3β-{[(amino)(imino)methyl)amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]-cyclopentancarboxylicacid;

cis-3-{[(amino)(imino)methyl]amino)-trans-1-hydroxy-trans-4-[(1-methylcarbonylamino)(2-trifluoromethyl-carbonyloxy)ethyl]cyclopentan-r-carboxylicacid;

t-3-amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-hydroxycyclopentan-r-carboxylicacid;

c-3-{[(amino(imino)methyl]amino}-t-1-hydroxy-t-4-{(methylcarbonylamino)([(methyl)-(methoxy)amino]carbonyl}methyl}cyclopentan-r-carboxylicacid;

3β-{[(amino)(imino)methyl]amino}-4α-{{4-[(methoxy)(methyl)amino]1-(methylcarbonylamino-2-oxo}butyl}cyclopentancarboxylicacid;

t-3-{[(amino)(imino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid;

t-3-amino-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxy-cyclopentan-r-carboxylicacid;

t-3-{[(amino)(imino)methyl]amino}-c-4-[di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-hydroxycyclopentan-r-carboxylicacid;

c-3-{[(amino)(imino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid;

3β-{[(amino)(imino)methyl]amino}-4α-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl)cyclopentancarboxylicacid;

3β-{[(amino)(imino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]cyclopentancarboxylicacid;

3β-{[Amino)(imino)methyl]amino}-4α-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]cyclopentancarboxylicacid;

3β-{1[(Amino)(imino)methyl]amino}-4α-{[(ethyl)(propyl)aminocarbonyl](methyl-carbonylamino)methyl}cyclopentancarboxylicacid;

3β-{([(Amino)(imino)methyl]amino}-4α-{[(ethyl)(propyl)aminocarbonyl](methyl-carbonylamino)methyl}cyclopentancarboxylicacid;

3β-{[(Amino)(imino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclopentancarboxylicacid;

3β-{[(Amino)(imino)methyl]amino}-4α-[1-(-methylcarbonylamino)pentyl]cyclopentancarboxylicacid.

In addition, an exemplary key intermediate, methyl3-t-butoxycarbonylamino-4-formylcyclopentanecarboxylate 6 (Scheme 4),may be synthesized from methyl3-hydroxy-4-hydroxymethylcyclopentanecarboxylate 1 (synthesis given inthe attached sheets). The primary hydroxyl of 1 may be protected withthe TBDMS (tert-butyldimethylsilyl) group; secondary hydroxyl groupsupon Mitsunobu reaction (Ph₃P, DEAD (diethyl azodi carboxylate), N₃H)can give the azido 3; azido 3 is reduced (H₂, Pd/C in presence of((t-boc)₂O) to give protected amine 4; primary hydroxyl may bedeprotected and on oxidation may give the key intermediate aldehyde 6.

As shown in Scheme 5, the aldehyde 6 may be further coupled with anappropriate allyl or vinyl tributyl tin compound to introduce the moietyfor the glycol or glycerol side chain. The scheme has been elaboratedwith vinyl tributyl tin. The t-boc group in compound 7 may be removed(trifluoroacetic acid) to an amine 8 and the amine may be reacted withbis boc (—OC(═O)C(CH₃)₃) thiourea to give the protected guanidine 9. Thehydroxyl of 9 upon Mitsunobu reaction can give azide 10; the azide canbe reduced to amine 11, and further acylated with an appropriate alkylacid or alkyl sulfonyl chloride to give the desired 12. The double bondof an allyl or vinyl group in the side chain on osmium catalyseddihydroxylation could give compound 13, which upon further deprotectionscould yield the desired target 14.

Before the deprotection stage in compound 13, the primary hydroxyl maybe converted to a tosyl (4—CH₃ phenyl SO₂) group, conversion of a tosylto an azide and an azide to an amine give the compounds whereR₃═CH(OH)CH₂NH₂, CH(OH)CH(OH)CH₂NH₂, or CH₂CH(OH)CH₂NH₂.

The synthetic route to prepare the compounds, when R₇ is OH, CN, NH₂ orNO₂ is shown in Scheme 6. The amine 8 upon reaction with cyanogenbromide could give the cyanamine 15; the side chain may be manipulatedin the same manner as shown in Scheme 5 to give 16; the further reactionof cyanamine 16 with hydroxylamine hydrochloride, hydrazine or cyanamidecould give the appropriate 17, which on deprotection may yield thetargets 18.

The reaction of 8 with 2-methyl-1-nitro-2-thiopseudourea leads to 18(R₇═NO₂). When R₂ is

the compounds of the type 12 on reaction with P₂S₅ or Lawsson's reagent(2,4-bis(4-methoxy phenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide)could give compound 19, which on further reactions may be converted tothe desired targets.

Other methods to prepare the equivalent of key intermediate 5 arepresented in Schemes 8 and 9. The intermediate 25 may be prepared by twodifferent procedures:

i) Scheme 8

The reaction of dimethylmalonate with sodium hydride and then cis1,4-dichloro-2-butene gives 1,1-dimethyl-3-cyclopentene dicarboxylate20, which is saponified, decarboxylated and esterified to give thebenzyl ester of 3-cyclopentene 22. Compound 22 upon reaction withPhI═NTS give aziridine 23. The aziridine may be opened withbisphenylthiomethane and n-butyllithium to give 24, and 24 upon reactionwith copper oxide and copper chloride could yield 25. Compound 25 may beused in Scheme 5 and elaborated in the same manner as 5 to give thedesired targets.

ii) Scheme 9

The cyclopentene ester 22 upon an hydroxyamination reaction withchloramine-T in the presence of OsO₄ gives 26; the isomers areseparated. The desired isomer upon reaction with 4-nitrobenzenesulfonylchloride gives 27, and the ONS group may be displaced withbisphenylthiomethane to give 24. Conversion to 25 occurs as describedabove.

The following Scheme 11 illustrates a procedure for preparing compoundsof Examples 6, 7, 20, 26, 27, 28, and 29 represented by the formula:

EXAMPLE

6 R=guanidine; R′=CH₂OH isomer A at C-6

7 R=guanidine; R′=CH₂OH isomer B at C-6

20 R=guanidine;

26 R=guanidine;

27 R=guanidine;

28 R=guanidine;

isomer A

29 R=guanidine;

isomer B

The following scheme 12A or scheme 12B illustrates a procedure forpreparing compounds of examples 16, 17, 19, 24, and 25 represented bythe formula:

EXAMPLE

16 R=guanidine,

17 R=guanidine,

19 R=guanidine,

24 R=guanidine,

25 R=guanidine,

F˜Compounds of Examples 6 and 7

F˜Compounds of Examples 26, 27, 28 and 29

F˜the Compound of Example 20

The following Scheme 13 illustrates a procedure for preparing compoundsof Examples 8, 9, 18, 21, 22 and 23 represented by the followingformula:

EXAMPLE

8 R=NH₂, R′=CH₂OH

9 R=guanidine, R′=CH₂OH

18 R=NH2, R′=CH₂OCH₂C₆H₅

21 R=guanidine,

22 R=NH₂,

23 R=guanidine,

The following Scheme 14 illustrates a procedure for preparing compoundsof Examples 10, 11, 12, 13, 14, and 15 represented by the formula:

The following non-limiting Examples are presented to further illustratethe present invention.

Example 1

cis-3-[(methylcarbonylamino)methyl]cyclopentancarboxylic acid

To a mixture of cis-3-(methoxycarbonyl)cyclopentancarboxylic acid[prepared according to the procedure disclosed by Tucjan J. J.Horonowski and Walter A. Szorek. Can. J. Chem. 66 61-70 (1988); 17.2 g,0.1 mmol] and triethylamine (10.1 g, 0.1 mmol) in tetrahydrofuran (300mL) was added ethyl chloroformate (10.9 g, 0.1 mmol) at about −5° C.over a period of about 30 min. The mixture was further stirred for about15 min at about −5° C. The resultant thick slurry was filtered and thesolids were washed with tetrahydrofuran. The combined filtrates werecooled to about 10° C. and sodium borohydride (14.5 g, 0.38 mol) wasadded with stirring in one portion. The mixture was stirred further forabout 10 min and methanol (62 mL) added dropwise over a period of about1 h at about 10° C. When methanol addition was completed, 6 Nhydrochloric acid was added slowly until neutralization. An organiclayer was collected and an aqueous layer was extracted thrice withether. The combined organic extracts were dried, filtered, andconcentrated. The residue was dissolved in ether (about 300 mL), and awhite solid was precipitated which was removed by filtration. Thefiltrate was concentrated and the residue passed through a column ofsilica gel using about 2% methanol in chloroform. The appropriatefractions were combined and concentrated to give 12.5 g (79%) of methylcis-3-hydroxymethylcyclopentancarboxylate.

To a mixture of methyl cis-3-hydroxymethylcyclopentancarboxylate (4.74g, 30 mmol) in dry benzene (about 250 mL) was added triphenyl phosphine(7.86 g, 30 mmol), diethyl azodicarboxylate (5.22 g, 30 mmol) andhydrazoic acid (2.5 M in toluene, 14.0 mL, 35 mmol). The mixture wasstirred at room temperature for about 16 h. The reaction mixture wasconcentrated and the residue was dissolved in ethyl acetate and hexanewas added. A precipitate formed on standing in about 2 h and was removedby filtration. The filtrate was concentrated and the residue was passedthrough a column of silica gel using chloroform as eluent. Theappropriate fractions were combined and concentrated to give 2.6 g (47%)of methyl cis-3-azidomethyl-cyclopentancarboxylate.

A mixture of methyl cis-3-azidomethylcyclopentancarboxylate (1.2 g, 6.5mmol) and 1 N sodium hydroxide (12.0 mL, 12 mmol) was stirred at roomtemperature for about 8 h. The alkaline mixture was extracted withchloroform. The aqueous layer was acidified with hydrochloric acid andextracted twice with chloroform. Combined chloroform extracts from theacidic mixture were dried over sodium sulfate, filtered and concentratedto give about 1.0 g (91%) of cis-3-azidomethylcyclopentancarboxylicacid.

Analysis: Calculated for C₇H₁₁N₃O₂: C, 49.70; H, 6.55; N, 24.84 Found:C, 49.92; H, 6.49; N, 24.58

A mixture of cis-3-azidomethylcyclopentancarboxylic acid (0.9 g, 5.3mmol) in methanol (about 30 mL) was hydrogenated at about 50 psi androom temperature in the presence of 10% Pd on carbon (about 40 mg) forabout 1 h. The catalyst was removed by filtration and the filtrateconcentrated to give a syrup. When the syrup was dissolved indimethylformamide and dichloromethane for acetylation (next step),formation of some white precipitate occurred. The precipitate wascollected by filtration, washed with dichloromethane and dried to givecis-3-aminomethylcyclopentanecarboxylic acid as a white powder, mp186-188° C. The yield of syrup was 0.7 g (100%). The white solidobtained was about 0.35 g. The remainder of the material from thedimethylformamide and dichloromethane mixture was not recovered, but wasused for the acetylation step.

Analysis: Calculated for C₇H₁₃NO₂.0.1H₂O: C, 57.99; H, 9.18; N, 9.66Found: C, 57.70; H, 9.01; N, 9.50

To a mixture of cis-3-aminomethylcyclopentancarboxylic acid (0.2 g, 1.4mmol) in pyridine (about 5 mL) was added acetic anhydride (about 1 mL)and the mixture stirred at about 70° C. for about 2 h. The solvent wasremoved under reduced pressure, water was added several times andevaporated in vacuo. A light, viscous oil was obtained and dried invacuo over acetone reflux to give 0.25 g (97%) ofcis-3-[(methylcarbonylamino)methyl]cyclopentancarboxylic acid.

Analysis: Calculated for C₉H₁₅NO₃: C, 58.36; H, 8.16; N, 7.56 Found: C,58.90; H, 7.94; N, 6.90

Example 2

t-3-amino-c-4-(methylcarbonylamino)methyl-r-cyclopentancarbonylic acidtrifluoroacetic acid [1:1]

To a mixture ofc-2-acetyloxy-c-4-methoxycarbonyl-r-cyclopentancarboxylic acid [preparedaccording to the procedure disclosed by Tucjan J. J. Horonowski andWalter A. Szorek, Can. J. Chem. 63 2787-2797 (1985); 23.4 g, 101.64mmol] in tetrahydrofuran (about 250 mL) and triethylamine (about 14.96mL, 106.7 mmol), ethyl chloroformate (about 10.2 mL, 106.7 mmol) wasadded over a period of about 15 min at about 0° C. After stirring for anadditional 30 min at the same temperature, the mixture was filtered withsuction, and the cake washed with tetrahydrofuran (3×10 mL).

Sodium borohydride (15.7 g, 406.56 mmol) was added to the filtrate inone portion. Then methanol (about 64 mL) was added dropwise over aperiod of about one hour at about 10° C. After stirring for additional30 min the reaction mixture was quenched carefully with 1 N HCl to pH 7.The solvent was removed in vacuo to obtain a white solid. The solid wasdissolved in water (about 50 mL) and HCl (1 N) is added to adjust the pHto about 6. The solution was homogeneous. The solution was extractedwith ethyl acetate (4×150 mL). The organic layers are combined andwashed with brine (about 150 mL) and dried with sodium sulfate. Thesolvent was removed in vacuo to give a white solid. The white solid wasdissolved in ether (about 200 mL) and filtered to remove insolubleimpurities. The filtrate was concentrated in vacuo to furnish 21.5 g(97%) of a mixture of methylc-4-methylcarbonyoxymethyl-c-3-hydroxycyclopentancarboxylate, methylc-3-methylcarbonyloxy-c-4-hydroxymethylcyclopentan-r-carboxylate, andmethyl c-3-hydroxy-c-4-hydroxymethylcyclopentan-r-carboxylate.

The above mixture was dissolved in methanol (about 200 mL) and sodiummethoxide solution (about 7 mL, 25% wt in methanol) was added dropwiseat room temperature. The reaction mixture ws stirred for about 90 minand solvent was removed in vacuo. The white residue was purified byflash column chromatography {20-40% [chloroform/methanol/conc ammoniumhydroxide (80:18:2)] in dichloromethane} to furnish 13.2 g (76%) of puremethyl c-3-hydroxy-c-4-hydroxymethylcyclopentan-r-carboxylate as a whitesolid.

To methyl c-3-hydroxy-c-4-hydroxymethylcyclopentan-r-carboxylate (13.2g, 76.3 mmol), triphenylphosphine (44.5 g, 167.86 mmol) and diethylazodicarboxylate (27.82 mL, 167.86 mmol) in anhydrous benzene (about 800mL) was added dropwise with stirring under nitrogen at room temperaturehydrazoic acid (1 M solution in toluene, 190.75 mL, 190.75 mmol) over aperiod of about 30 min. The reaction mixture was stirred overnight atroom temperature and concentrated in vacuo to half the original volume.The solid obtained on standing was removed by filtration and thefiltrate was concentrated in vacuo to furnish an orange residue. Theresidue was crystallized from ethyl acetate/hexane to removetriphenylphosphine oxide and 1,2-ethoxycarbonylhydrazine. The filtrateis concentrated in vacuo and purified by flash column chromatography(5-25% ethyl acetate in hexane) to furnish 8.8 g (52%) of methylt-3-azido-c-4-azidomethylcyclopentan-r-carboxylate.

To methyl t-3-azido-c-4-azidomethylcyclopentan-r-carboxylate (about 8.4g, 37.5 mmol) was added 1 N sodium hydroxide (112.5 mL, 112.5 mmol) andstirred overnight at room temperature. The reaction mixture wasextracted with chloroform (3×20 mL). The aqueous layer was acidified topH 4 using conc HCl and extracted with chloroform (4×25 mL). The organiclayers were combined, dried with magnesium sulfate, filtered through apad of silica and concentrated in vacuo to obtain 7.58 g (96%) oft-3-azido-c-4-azidomethyl-c-cyclopentan-r-carboxylic acid as a colorlessoil.

Analysis: Calculated for C₇H₁₀N₆O₂: C, 40.00; H, 4.80; N, 39.98 Found:C, 40.24; H, 4.88; N, 39.73

To a solution of t-3-azido-c-4-azidomethylcyclopentan-r-carboxylic acid(1.05 g, 5.0 mmol), dicyclohexylcarbodiimide (2.5 g, 12 mmol) and4-dimethylaminopyridine (60 mg, 0.5 mmol) in dichloromethane (about 25mL), was added dropwise over a period of about 15 min at roomtemperature tert-butanol (0.97 mL, 10.0 mmol). After stirring at roomtemperature overnight, the mixture was filtered with suction and thecake washed with ether (3×5 mL). The filtrate was concentrated in vacuoand ether (about 25 mL) was added to the residue. The organic layer waswashed with cold HCl (1%, 20 mL), saturated sodium bicarbonate (2×20mL), water (20 mL), dried with magnesium sulfate, filtered and solventremoved in vacuo to obtain 2.92 g of an oil. Purification of the crudeby flash column chromatography (2-5% ether in hexane) provided 0.39 g(29%) of t-3-azido-c-4-azidomethyl-r-1-tert-butoxycarbonylcyclopentaneas a colorless oil.

Analysis: Calculated for C₁₁H₁₈N₆O₂: C, 49.61; H, 6.81; N, 31.56 Found:C, 49.83; H, 6.76, N, 31.38

To a solution oft-3-azido-c-azidomethyl-r-1-tert-butoxycarbonylcyclopentane (0.33 g,1.22 mmol) in methanol (about 10 mL) was added under nitrogen Pd/C (0.1g, 10% Palladium content) and the resulting mixture hydrogenated atabout 50 psi for about 20 min. The hydrogen was evacuated, and afteraddition of fresh hydrogen, the resulting mixture was hydrogenated atabout 50 psi for about 40 min. The catalyst was removed by filtrationthrough Celite. The filtrate was concentrated in vacuo to furnish about0.25 g (97%) oft-3-amino-c-4-aminomethyl-r-1-tert-butoxycarbonylcyclopentane.

To a solution oft-3-amino-c-4-aminomethyl-r-1-tert-butoxycarbonylcyclopentane (0.25 g,1.22 mmol), in dichloromethane (about 10 mL), cooled to 0° C. is addeddropwise with stirring over a period of about 5 min acetic anhydride(0.098 mL, 1.04 mmol). After stirring at 0° C. for about one h, thereaction mixture was stirred at room temperature overnight. The solventwas removed in vacuo to obtain a white solid. The crude is purified byflash column chromatography {30-100% [chloroform/methanol/conc ammoniumhydroxide(80:18:2)] in dichloromethane} and furnished:

1.t-3-methylcarbonylamino-c-4-(methylcarbonylamino)methyl-r-tert-butoxycarbonyl-cyclopentane,0.04 g (11%) as a white solid, mp 170-172° C.

Analysis: Calculated for C₁₅H₂₆N₂O₄.0.25H₂O: C, 59.48; H, 8.82: N, 9.25Found: C, 59.59; H, 8.82; N, 9.17

2.t-3-amino-c-4-(methylcarbonylamino)methyl-r-1-tert-butoxycarbonylcyclopentane,0.12 g (39%) as a white solid, mp 86-87° C.

Analysis: Calculated for C₁₃H₂₄N₂O₃: C, 60.91; H, 9.44; N, 10.93 Found:C, 60.69; H, 9.40; N, 10.87

3. t-3-amino-c-4-aminomethyl-r-1-tert-butoxycarbonylcyclopentane, 0.05 g(20%) as a colorless oil. The oil was dissolved in dichloromethane (2mL) and acetic acid (3 equi) was added. The resulting solution wasstirred for 15 min and the solid obtained was filtered to obtain theproduct as an acetate, mp 124-127° C.

Analysis: Calculated for C₁₁H₂₂N₂O₂.2C₂H₄O₂.0.25H₂O: C, 53.16; H, 9.07;N, 8.27 Found: C, 53.34; H, 8.81; N, 8.46

To a solution oft-3-amino-c-4-(methylcarbonylamino)methyl-r-1-tert-butoxycarbonyl-cyclopentane(0.09 g, 0.32 mmol) in dichloromethane (about 3 mL) was added dropwisetrifluoroacetic acid (about 0.67 mL, 8.8 mmol) and stirred at roomtemperature for about 2 h. The solvent was removed in vacuo and theexcess trifluoroacetic acid was removed by co-distilling thrice in vacuowith dichloromethane (about 5 mL) to obtain 0.1 g (99%) oft-3-amino-c-4-[(methylcarbonylamino)methyl]-cyclopentan-r-carboxylicacid trifluoroacetic acid as a hydroscopic, tan solid.

Analysis: Calculated for C₉H₁₆N₂O₃.CF₃CO₂H: C, 42.04; H, 5.45; N, 8.90Found: C, 41.75; H, 5.58; N, 8.59

Example 3

t-3-{[(Amino)(imino)methyl]amino}-c-4-[(methylcarbonylamino)methyl]-cyclopentan-r-carboxylicacid trifluoroacetic acid [1:1.25]

To a stirred solution oft-3-azido-c-4-azidomethylcyclopentan-r-carboxylic acid from Example 2(about 4.0 g, 19.05 mmol) in dichloromethane (about 40 mL) was rapidlyadded liquefied isobutylene (about 20 mL), followed by the dropwiseaddition of phosphoric acid (prepared by saturating 2.5 mL of 85% H₃PO₄with P₂O₅) in dichloromethane (2.5 mL) and boron trifluonde etherate(0.9 mL). After the mixture was stirred at about −78° C. for about 2 hat room temperature overnight, ice-water and saturated aqueous NaHCO₃was added until the mixture becomes basic. The aqueous layer wasextracted with dichloromethane (2×20 mL). The organic layers werecombined, washed with water (20 mL) and brine (20 mL), dried overanhydrous MgSO₄, and concentrated in vacuo to furnish 1.7 g of acolorless oil. Purification of the crude by flash column chromatography(5-10% ether in hexane) gave 1.2 g (24%) oft-3-azido-c-4-azidomethyl-r-1-tert-butoxycarbonylcyclopentane.

To a solution oft-3-azido-c-4-azidomethyl-r-1-tert-butoxycarbonylcyclopentane (1.22 g,4.6 mmol) in methanol (20 mL) was added under nitrogen Pd/C (0.1 g, 10%Palladium content) and the resulting mixture hydrogenated at about 50psi for about 30 min. The hydrogen was evacuated, and after addition offresh hydrogen, the resulting mixture was hydrogenated at about 50 psifor about 30 min. The catalyst was removed by filtration through Celite.The filtrate was concentrated in vacuo to furnish about 0.94 g (96%) oft-3-amino-c-4-aminomethyl-r-1-tert-butoxycarbonylcyclopentane.

To a solution oft-3-amino-c-4-aminomethyl-r-1-tert-butoxycarbonylcyclopentane (0.63 g,2.95 mmol), in dichloromethane (30 mL), cooled to about −5° C. aceticanhydride (0.25 mL, 2.65 mmol) was added dropwise with stirring over aperiod of about 5 min. After stirring at 0° C. for about one hour thereaction mixture was stirred at room temperature overnight. The solventwas removed in vacuo to obtain a white solid. Purification of the crudeby flash column chromatography {30-100% [chloroform/methanol/concammoniumhydroxide (80:18:2)] in dichloromethane} gives 0.33 g (44%) oft-3-amino-r-1-tert-butoxycarbonyl-c-4-(methylcarbonylamino)methylcyclopentane.

Tot-3-amino-r-1-tert-butoxycarbonyl-c-4-(methylcarbonylamino)methylcyclopentane(0.33 g, 1.29 mmol) in dimethyl formamide (about 5 mL) was addedtriethylamine (0.63 mL, 4.52 mmol) and bis-bocthiourea (0.4 g, 1.42mmol). The resulting mixture was cooled to 0° C. and mercuric chloride(0.39 g, 1.42 mmol) was added. The reaction mixture was stirred at 0° C.for about 30 min and then at room temperature overnight. Ethyl acetate(about 50 mL) was added and the slurry filtered through Celite. Thefiltrate was washed with water (2×20 mL) and brine (20 mL), the organiclayer was dried (MgSO₄), filtered, and solvent removed in vacuo to giveabout 0.7 g of crude. The crude was purified by flash columnchromatography (30-50% ethyl acetate in hexane) and recrystallized fromether/hexane to obtain 0.51 g (79%) of a colorless oil. The oil wascrystallized from petroleum ether to obtaint-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-cis-1-tert-butoxycarbonyl-c-4-(methylcarbonylamino)-methyl]cyclopentaneas a white solid, mp 145-146° C.

Analysis: Calculated for C₂₄H₄₂N₄O₇: C, 57.81; H, 8.49; N, 11.24 Found:C, 57.70; H, 8.52; N, 10.98

To a solution oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-cis-1-tert-butoxycarbonyl-c-4-[(methylcarbonylamino)methyl]cyclopentane(0.44 g, 0.88 mmol) in dichloromethane (about 9 mL) is added dropwisetrifluoroacetic acid (1.7 mL, 22.1 mmol) and stirred at room temperaturefor about 1 h. The solvent was removed in vacuo and the excesstrifluoroacetic acid was removed by co-distilling thrice in vacuo withdichloromethane (about 5 mL) to obtain a white solid. The white solidwas triturated with ether and dried in vacuo at acetone reflux to obtain0.21 g (67%) oft-3-{[(amino)(imino)methyl]amino}-c-4-[(methylcarbonylamino)methyl)cyclopentan-r-carboxylicacid trifluoroacetic acid as a hygroscopic solid.

Analysis: Calculated for C₉H₁₆,N₂O₃.1.25CF₃CO₂H: C, 39.30; H. 5.04; N.14.56 Found: C; 39.21; H, 5.29; N, 14.26

Example 4

c-3-{{[(Amino)(imino)methyl]amino}methyl}cyclopentan-r-carboxylic acid

To a mixture of c-3-aminomethylcyclopentancarboxylic acid from Example 1(0.07 g, 0.5 mmol) and potassium carbonate (0.07 g, 0.5 mmol) in water(about 1.5 mL) was added aminoiminomethane sulphonic acid (0.06 g, 0.5mmol) and the mixture was stirred for about 18 h at room temperature. Awhite solid separated, which was collected by filtration, washed with asmall amount of water and dried in vacuo to give 0.04 g (49%) ofc-3-{{[(amino)-(imino)methyl]amino}methyl}cyclopentancarboxylic acid aswhite powder, mp 280° C. (darkens).

Analysis: Calculated for C₃H₁₅N₃O₂: C, 51.88; H, 8.16; N, 22.69 Found:C, 51.79; H, 8.09; N, 22.63

Example 5

t-3-{[(Amino)(imino)methyl]amino}-c-4-{{[(amino)(imino)methyl]amino}methyl}cyclopentan-r-carboxylicacid dihydrochloride hydrate 2-propanol (5:10:5:2)

To a stirred solution oft-3-azido-c-4-azidomethylcyclopentan-r-carboxylic acid from Example 2(about 4.0 g, 19.05 mmol) in dichloromethane (about 40 mL) was rapidlyadded liquefied isobutylene (about 20 mL), followed by the dropwiseaddition of phosphoric acid (prepared by saturating 2.5 mL of 85% H₃PO₄with P₂O₅) in dichloromethane (2.5 mL) and boron trifluoride etherate(0.9 mL). After the mixture was stirred at about −78° C. for about 2 hat room temperature overnight, ice-water and saturated aqueous NaHCO₃was added until the mixture becomes basic. The aqueous layer wasextracted with dichloromethane (2×20 mL). The organic layers werecombined, washed with water (20 mL) and brine (20 mL), dried overanhydrous MgSO₄, and concentrated in vacuo to furnish about 1.7 g of acolorless oil. Purification of the crude by flash column chromatography(5-10% ether in hexane) gave 1.2 g (24%) oft-3-azido-c-4-azidomethyl-r-1-tert-butoxycarbonylcyclopentane.

To a solution oft-3-azido-c-4-azidomethyl-r-1-tert-butoxycarbonylcyclopentane (1.22 g,4.6 mmol) in methanol (about 20 mL) was added under nitrogen Pd/C (0.1g, 10% Palladium content) and the resulting mixture hydrogenated atabout 50 psi for about 30 min. The hydrogen was evacuated, and afteraddition of fresh hydrogen, the resulting mixture was hydrogenated atabout 50 psi for about 30 min. The catalyst was removed by filtrationthrough Celite. The filtrate was concentrated in vacuo to furnish about0.94 g (96%) oft-3-amino-c-4-aminomethyl-r-tert-butoxycarbonylcyclopentane.

To a solution oft-3-amino-c-4-aminomethyl-r-tert-butoxycarbonylcyclopentane (0.32 g, 1.5mmol) in dimethylformamide (about 5 mL) was added triethylamine (1.5 mL,10.5 mmol) and bis-bocthiourea (0.91 g, 3.3 mmol). The resulting mixturewas cooled to 0° C. and mercuric chloride (0.9 g, 3.3 mmol) was added.The reaction mixture was stirred at 0° C. for about 30 min and then atroom temperature overnight. Ethyl acetate (about 25 mL) was added andthe slurry filtered through Celite. The filtrate was washed with water(2×20 mL) and brine (20 mL), the organic layer dried (MgSO₄) filteredand solvent removed in vacuo to give about 1.22 g of crude. The crude ispurified by flash column chromatography (25-36% ether in hexane) tofurnish 0.75 g (71%) of a colorless oil. The oil was recrystallized frompetroleum ether to obtaint-3-{(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-{{tert-butoxycarbonyl-amino)(tert-butoxycarbonylimino)methyl]amino}methyl}-r-tert-butoxycarbonylcyclopentaneas a white solid, mp 152-154° C.

Analysis: Calculated for C₃₃H₅₈N₆O₁₀: C, 56.72; H, 8.37; N, 12.03 Found:C, 56.96; H, 8.51; N, 12.06

A solution oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-{{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}methyl}-r-tert-butoxycarbonylcyclopentane(0.56 g, 0.88 mmol) in 5 N hydrochloric acid (about 1 mL) was stirred atroom temperature for about 2 h. The solvent was removed in vacuo tofurnish a hygroscopic residue. The residue was washed several times withether and dried in vacuo at acetone reflux temperature to obtain 0.18 g(64%) oft-3-{[(amino)(imino)methyl]amino}-c-4-{{[(amino)(imino)methyl]amino}methyl}cyclopentan-r-carboxylicacid dihydrochloride hydrate 2-propanol (5:10:5:2) as a hygroscopicsolid.

Analysis: Calculated for C₉H₁₆N₂O₃.2HCl.H₂O.0.4C₃H₈O: C; 34.29; H, 7.11;N, 23.52 Found: C; 34.38; H, 6.84; N, 23.94

Example 6

F.W. 272.30

4β-{[(Amino)(imino)methyl]amino}-3α-[(2-hydroxy-1-methylcarbonylamino)ethyl]-1-cyclo-pentancarboxylicacid (isomer A at C-6)

To a solution of sodium azide (2.12 g, 32.6 mmol) in dimethylformamide(15 mL) cooled to 0° C. was added dropwise with stirring4-bromocyclopent-2-enone (DePuy, C. H.; Isaks, M.; Eilers, K. L.;Morris, G. F. J. Org. Chem. 1964. 29, 3503; 3.5 g, 21.7 mmol) indimethyl-formamide (5 mL) over a period of 5 min. The reaction mixturewas stirred at 0° C. for 30 min and diluted with ethyl acetate (20 mL).The reaction mixture was washed with water (2×20 mL) and brine (20 mL),dried (MgSO₄), filtered, and concentrated in vacuo to furnish an oilyresidue. Purification of the crude oil by flash column chromatography(10-15% ethyl acetate in hexane) gave 1.9 g (71%) of4-azidocyclopent-2-enone as a light-yellow oil.

To a solution of diethyl acetamidomalonate (Aldrich, 1.25 g, 5.7 mmol)in ethanol (10 mL) under nitrogen was added freshly cut sodium metal(0.03 g, 1.4 mmol). The reaction mixture was stirred at room temperatureuntil all sodium has dissolved. The reaction mixture was cooled to −40°C. (dry ice/acetonitrile) and a solution of 4-azidocyclopent-2-enone(0.7 g, 5.7 mmol) in ethanol (5 mL) was added dropwise over a period of10 min. The reaction mixture was stirred at −40° C. for 30 min andquenched with trifluoroacetic acid (0.1 mL, 1.4 mmol). The solvent wasremoved in vacuo to furnish a white solid. Purification of the crude byflash column chromatography (60% ether in hexane) gave 1.2 g (63%) oftrans-3-azido-4-[(methylcarbonyl-amino)bis(ethoxycarbonyl)methyl]cyclopentanoneas a white solid, mp 121-122° C.

Analysis: Calculated for C₁₄H₂₀N₄O₆: C, 49.41; H, 5.92; N, 16.46 Found:C, 49.47; H, 5.95; N, 16.48

To a mixture oftrans-3-azido-4-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]-cyclopentanone(8.2 g, 24.1 mmol) in methanol (100 mL) was added sodium borohydride(0.46 g, 12.1 mmol) in portions over a period of 5 min at roomtemperature. The reaction mixture was further stirred for 10 min andthen acetic acid (1 mL) was added and mixture concentrated. The residuewas dissolved in ethyl acetate (100 mL) and washed with water (1×100 mL)and brine (1×100 mL) and dried (MgSO₄). After filtration, the filtratewas concentrated to give 8.2 g (100%) of3β-azido-4α-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentanol,a mixture of isomers, as a light-brown oil.

Analysis: Calculated for C₁₄H₂₂N₄O₆: C, 49.12; H, 6.48; N, 16.37 Found:C, 49.16; H, 6.51; N, 16.11

A mixture of3β-azido-4α-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclo-pentanol,a mixture of isomers (8.0 g, 23.4 mmol), and sodium hydroxide (1 N, 72.0mL, 72.0 mmol) was stirred for 16 h at room temperature. Acetic acid(glacial, 20 mL) was added and heated at gentle reflux for 2 h. Themixture was then extracted with ethyl acetate (3×100 mL). The combinedorganic layers were washed with water (2×100 mL) and brine (1×100 mL)and dried (MgSO₄). After filtration, the filtrate was concentrated andthe residue passed through a column of silica gel using ethyl acetate asan eluent to give 4.0 g (63%) of3β-azido-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentanol,a mixture of isomers, as a colorless syrup.

Analysis: Calculated for C₁₁H₁₈N₄O₄: C, 48.89; H, 6.71; N, 20.72 Found:C, 48.76; H, 6.72; N, 20.65

A mixture of the above alcohol, a mixture of isomers (3.8 g, 14.0 mmol),in ethyl acetate (120 mL) was hydrogenated in the presence of Pd/C (250mg, 10% Palladium content) at 40 psi and room temperature for 16 h. Thecatalyst was removed by filtration and the filtrate concentrated to give2.8 g (82%) of3β-amino-4α-[(ethoxycarbonyl)(methylcarbonylamino)-methyl]cyclopentanolas a syrup.

To a mixture of3β-amino-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclo-pentanol(2.8 g, 11.5 mmol) in dimethylformamide (25 mL) was added triethylamine(4.06 g, 40.02 mmol). The mixture was cooled in an ice bath andbis-bocthiourea (3.17 g, 11.5 mmol) was added, stirred for 10 min andmercury(II) chloride (3.12 g, 11.5 mmol) added. The reaction mixture wasstirred at ice bath temperature for 1 h, diluted with ethyl acetate (100mL), and filtered through Celite. The filtrate was washed with water(1×100 mL) and brine (1×100 mL) and dried (MgSO₄). After filtration, thefiltrate was concentrated and the residue passed through a column ofsilica gel (100 g) using ethyl acetate as an eluent to give 5.0 g (89%)of3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino)-4α-[(ethoxycarbonyl)(methyl-carbonylamino)methyl]cyclopentanol,a mixture of isomers, as a white foam. An analytical sample was preparedby recrystallization from ethyl acetate/hexane.

Analysis: Calculated for C₂₂H₃₈N₄O₈: C, 54.31; H, 7.87: N, 11.51 Found:C, 54.47; H, 7.95; N, 11.39

To a mixture of3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentanol(4.02 g, 8.27 mmol) in dichloromethane (75 mL) was added pyridiniumchlorochromate (5.3 g, 24.8 mmol) and stirred at room temperature for 16h. The mixture was diluted with ether, the reaction filtered throughCelite, and the filtrate concentrated. The residue was passed through acolumn of silica gel (200 g) using ethyl acetate/hexane (1:1) as aneluent to give:

1. 0.62 g (15%) of isomer A, higher running spot on TLC in ethylacetate, as a white powder, mp 175° C. An analytical sample of3β-{[(tert-butoxycarbonyl-amino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methyl-carbonylamino)methyl]cyclopentanone,isomer A, was prepared by recrystallization from ethyl acetate/hexane.

Analysis: Calculated for C₂₂H₃₆N₄O₈: C, 54.53; H, 7.49; N, 11.56 Found:C, 54.50; H, 7.44; N, 11.43

2. 0.1 g (2.5%) of isomer B lower running spot on TLC in ethyl acetate,as a white powder, mp 139° C. An analytical sample of3β-[(tert-butoxycarbonyl-amino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methyl-carbonylamino)methyl]cyclopentanone,isomer B, was prepared by recrystallization from ethyl acetate/hexane.

Analysis: Calculated for C₂₂H₃₆N₄O₈: C, 54.53; H, 7.49; N, 11.56 Found:C, 53.94; H, 7.44; N, 11.26

To a mixture of 2-trimethylsilyl-1,3-dithiane (0.67 g, 3.5 mmol) intetrahydrofuran (6 mL) at 0° C. was added n-butyllithium (1.6 M, 2.5 mL,3.7 mmol), and the mixture stirred at 0° C. for 0.75 h. After themixture was cooled to −42° C., a mixture of4β-{[(tert-butoxycarbonyl-amino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)-methyl]cyclopentanone,mixture of isomers (0.24 g, 0.5 mmol), in tetrahydrofuran (4 mL) wasadded and the mixture further stirred at −42° C. for 2 h. The mixturewas then quenched with saturated aqueous ammonium chloride (2 mL) andwarmed to the room temperature. The organic layer was separated, water(5 mL) added to the aqueous phase and extracted with ethyl acetate (2×15mL). The combined organic layers were washed with water (1×20 mL), brine(1×20 mL) and dried (MgSO₄). After filtration, the filtrate wasconcentrated and the residue passed through a column of silica gel (50g) using ethyl acetate/hexane (1:2) as an eluent to give:

1. 0.15 g (50%) of isomer A, the higher running spot on TLC in ethylacetate/hexane (1:2), as a white powder, mp 190-191° C. An analyticalsample of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}4α-[(ethoxy-carbonyl)(methylcarbonylamino)methyl]cyclopentylidene}-1,3-dithiane,isomer A, was prepared by recrystallization from ethyl acetate/hexane.

Analysis: Calculated for C₂₆H₄₂N₄O₇S₂: C, 53.22; H, 7.21; N, 9.55 Found:C, 53.44; H, 7.27; N, 9.31

2. 0.06 g (20%) of isomer B, the lower running spot on TLC in ethylacetate/hexane (1:2), as a white powder, mp 185-186° C. An analyticalsample of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxy-carbonyl)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithiane,isomer B, was prepared by recrystallization from ethyl acetate/hexane.

Analysis: Calculated for C₂₆H₄₂N₄O₇S₂: C, 53.22; H, 7.21; N, 9.55 Found:C, 53.19; H, 7.20; N, 9.52

To a solution of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)-methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentylidene}-1,3-dithiane,isomer A (1.2 g; 2.0 mmol), in tetrahydrofuran (40 mL) was addeddropwise under nitrogen lithium borohydride (Aldrich, 2M solution intetrahydrofuran, 2.0 mL, 4.0 mmol) and lithium9-borabicyclo[3.3.1]nonane hydride (Aldrich, 1M solution intetrahydrofuran, 0.2 mL, 0.2 mmol) and the reaction mixture was stirredat room temperature overnight. An additional three portions of lithiumborohydride (2M solution in tetrahydrofuran, 2.0 mL, 4.0 mmol) andlithium 9-borabicyclo[3.3.1]nonane hydride (1 M solution intetrahydrofuran, 0.2 mL, 0.2 mmol) were added over a period of 36 h. Thereaction was quenched with 1 N sodium hydroxide (10 mL), brine (10 mL)and stirred for 5 min. The reaction was acidified to pH 4 using glacialacetic acid, ether (20 mL) was added and the aqueous layer wasseparated. The aqueous layer was extracted with ether (2×20 mL), theorganic layers were combined, dried and concentrated in vacuo to obtaincrude. The crude was purified on a Chromatotron (50-100% ethyl acetatein hexane) to furnish 0.34 g (31%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)ethyl]cyclopentylidene}-1,3-dithiane,isomer A, recrystallized from ether as a white solid, mp 209-210° C.

Analysis: Calculated for C₂₄H₄₀N₄O₆S₂: C, 52.92; H, 7.40; N, 10.29Found: C, 53.03; H, 7.30; N, 10.19

To a solution of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)-methyl]amino}-4α-[(2-hydroxy)(1-methlycarbonylamino)ethyl]cyclopentylidene}-1,3-dithiane,isomer A (0.23 g, 0.4 mmol), in methanol (6.7 mL) was added 6 N HCl(0.83 mL, 4.99 mmol) and stirred at room temperature until all startingmaterial had disappeared (TLC analysis, ethyl acetate, ˜3.0 h). Thesolvent was removed in vacuo (water bath temperature ˜40° C.) to furnishcrude3β-{[(amino)(imino)methyl]amino}-4α-[(2-hydroxy)(1-methycarbonylamino)ethyl]-1-[(3-mercaptopropyl)-thiocarbonyl]cyclopentane{MS (ES+1) 363.5 [100%, (M+1)]}.

To the above crude was added water (1.4 mL) and conc ammonium hydroxide(1.4 mL) and the reaction was stirred for 3 h at room temperature. Thesolvent was removed in vacuo to furnish 0.6 g of the title compoundmixed with salts, MS (ES+1) 272.4 [100%, (M+1)].

Example 7

Sodium3β-{[(amino)(imino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-cyclopentan-r-carboxylate:diethylether:hydrate(12:4:3), isomer B

To a solution of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)-methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentylidene}-1,3-dithianefrom Example 6, isomer A (6.74 g, 11.5 mmol), in ethanol (57.5 mL) andtetrahydrofuran (115 mL) was added 1 N sodium hydroxide (23 mL, 23 mmol)and water (35 mL). The reaction was stirred at room temperature for 5 h.Tetrahydrofuran was removed in vacuo and the aqueous layer was washedwith ethyl acetate (2×10 mL) and acidified to pH 5 with glacial aceticacid. The solid obtained was collected by filtration to furnish 5.95 g(93%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(carboxy)(methylcarbonyl-amino)methyl]-1-cyclopentylidene}-1,3-dithianeas a white solid.

Analysis: Calculated for C₂₄H₃₈N₄O₇S₂.0.5C₂H₆O.1H₂O: C, 50.07; H, 7.23;N, 9.34 Found: C, 50.28; H, 6.85; N, 9.04

To a solution of the above acid (1.12 g, 2.0 mmol) in tetrahydrofuran(20 mL) cooled to 0° C. was added dropwise with stirring triethylamine(0.32 mL, 2.2 mmol) and ethyl chloroformate (0.21 mL, 2.1 mmol). Thereaction mixture was stirred for 1 h at 0° C. The reaction was filteredthrough Celite and the cake was washed with tetrahydrofuran (2×5 mL).The filtrate was cooled to 0° C., sodium borohydride (powder, 0.23 g,6.0 mmol) was added and methanol (1.3 mL) was then added dropwise over aperiod of 1 h. The reaction mixture was quenched with brine (10 mL) andacidified with glacial acetic acid to pH 5. Ether (10 mL) was added andthe organic layer was separated. The aqueous layer was extracted withether (2×10 mL). The organic layers were combined, dried andconcentrated in vacuo to furnish 1.1 g (100%) of2-{-3β{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methyl-carbonylamino)]-1-cyclopentylidene}-1,3-dithiane.

To a solution of above solid (3.67 g, 6.75 mmol) in dimethylformamide(16 mL) was added tert-butyldimethylchloro silane (1.15 g, 7.4 mmol) andimidazole (0.92 g, 13.5 mmol). The reaction mixture was stirred at roomtemperature overnight, poured into water (20 mL) and extracted withether (3×20 mL). The organic layers were combined, washed with water (20mL) and brine (25 mL), dried, and the solvent was removed in vacuo tofurnish 4.4 g of a colorless oil. Purification of the crude by flashcolumn chromatography (15-25% ethyl acetate in hexane) gave:

1. Isomer A, 2.5 g (57%) as a white solid, mp 129-130° C. (R_(f)=0.36,25% ethyl acetate in hexane); MS (ES+) 444.9 [100%,(M+1)-tert-butyldimethylsilyl].

Analysis: Calculated for C₃₀H₅₄N₄O₅S₂Si: C, 54.68; H, 8.26; N, 8.50Found: C, 54.71; H, 8.05; N, 8.61

2. Isomer B, 1.21 g (27%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxy-carbonylimino)methyl]amino}-4α-[(2-tert-butyldimethylsilyloxy)(1-methycarbonylamino)ethyl]cyclopentylidene}-1,3-dithiane,isomer B, as a white solid mp 94-96° C., (R_(f)=0.28, 25% ethyl acetatein hexane); MS (ES+) 659.4 [100%, (M+1)].

Analysis: Calculated for C₃₀H₅₄N₄O₅S₂Si: C, 54.68; H, 8.26; N, 8.50Found: C, 55.06; H, 8.18; N, 8.40

To a solution of 1.21 g (27%) of the above isomer B (1.11 g, 1.68 mmol),in methanol (50.4 mL), was added 6 N HCl (4.2 mL, 25.2 mmol) and themixture was stirred at room temperature until all starting material haddisappeared (TLC analysis, ethyl acetate, ˜30 h). The solvent wasremoved in vacuo (water bath temperature ˜35° C.) to furnish crude3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methyl-carbonylamino)ethyl]-1-[(3-mercaptopropyl)thiocarbonyl]cyclopentane{MS (ES+1) 563.5 [100%, (M+1)], 463.5 [80%, (M+1)-tert-butoxycarbonyl],363.4 [50%, (M+1)-di-tert-butoxycarbonyl]}.

The above crude was dried in vacuo to remove trace amounts of water andthen dissolved in dichloromethane (25 mL). Trifluoroacetic acid (1.29mL, 16.8 mmol) was added and the mixture was stirred overnight at roomtemperature. The solvent was removed in vacuo to furnish a crude residueof3β-[(amino)(imino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)-ethyl]-1-[(3-mercaptopropyl)thiocarbonyl]cyclopentane{MS (ES+1) 363.4 [100%, (M+1)]}.

The above crude residue was dissolved in tetrahydrofuran (8.5 mL),methanol (4.2 mL) and 1 N sodium hydroxide (8.4 mL, 8.4 mmol) were addedand the reaction mixture was stirred for 35 min at room temperature.Tetrahydrofuran and methanol were removed in vacuo and the aqueous layerwas washed with ethyl acetate (2×10 mL). The aqueous layer was filteredand filtrate was acidified to pH 6.5 (1 N HCl ). The acidified aqueouslayer was washed with ethyl acetate (2×10 mL) and concentrated in vacuoto furnish 1.3 g of crude.

The above crude (1.0 g) was loaded on a silica gel column (50 g) andeluted with chloroform:methanol:conc ammonium hydroxide (5:4:1) (1000mL) to remove organic and inorganic impurities. The column was theneluted with 25% water in 2-propanol to furnish 0.21 g (43%) of an oil.The oil was triturated with ethanol/ether and ether (5×10 mL) to furnishthe title compound as a white solid, mp 65° C. (fuses) (R_(f)=0.36, 25%water in 2-propanol, TLC plate developed with KMnO₄ spray).

Analysis: Calculated for C₁₁H₁₉N₄NaO₄.0.35C₄H₁₀O.0.25H₂O: C, 45.86; H,7.14; N, 17.25 Found: C, 46.14; H, 7.50; N, 17.55

Example 8

t-3-Amino-t-1-hydroxy-c-4-[(hydroxymethyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid trifluoroacetic acid ammonium trifluoroacetate (1:1:0.5)

A mixture oftrans-3-azido-4-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]-cyclopentanone(from Example 6, 0.50 g, 1.5 mmol), di-tert-butyl dicarbonate (Aldrich,0.39 g, 1.77 mmol), and Pd/C, 10% (0.140 g) in ethyl acetate (25 mL) washydrogenated at 45 psi for 1 h. The catalyst was removed by filtrationand the filtrate was concentrated in vacuo to afford 0.69 g of crude.Purification by flash column chromatography (silica gel, 75% ethylacetate/hexanes) gave a semisolid. Recrystallization of the residue fromether/hexanes provided 0.275 g (45%) oftrans-3-tert-butyloxycarbonylamino-4-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclo-pentanoneas a white solid, mp 135-136° C.

Analysis: Calculated for C₁₉H₃₀N₂O₄: C, 55.06; H, 7.30; N, 6.76 Found:C, 54.63; H, 7.17; N, 6.74

To a stirred solution of bis-(phenylthio)methane (Aldrich, 1.12 g, 4.84mmol) tetrahydrofuran (10 mL) at 0° C. was added dropwise n-butyllithium(1.6M, 3.0 mL, 4.84 mmol). After 30 min of stirring, the reactionmixture was cooled to −78° C. andtrans-3-tert-butyloxycarbonylamino-4-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentanone(0.50 g, 1.2 mmol) in tetrahydrofuran (6 mL) was added dropwise. Thereaction mixture was stirred for 1 h then quenched with a saturatedaqueous solution of ammonium chloride (10 mL). The separated aqueouslayer was extracted with ether (4×10 mL). The combined organic extractswere washed with brine, dried (MgSO₄), filtered through Celite, andconcentrated in vacuo to provide 1.25 g of crude. Purification by flashcolumn chromatography (silica gel, 75 g, 25% ethyl acetate/hexanes) gave0.16 g (20%) ofc-3-(tert-butoxycarbonylamino)-t-4-[bis(ethoxy-carbonyl)(methylcarbonylamino)methyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-olas a white solid, mp 36-37° C.

Analysis:

Calculated for C₃₂H₄₂N₂O₈S₂: C, 59.42; H, 6.54; N, 4.33 Found: C, 59.64;H, 6.45; N, 3.94

A mixture ofc-3-(tert-butoxycarbonylamino)-t-4-[bis(ethoxycarbonyl)(methylcarbonyl-amino)methyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-ol(0.63 g, 0.97 mmol) and potassium hydroxide (1 N, 3.4 mL, 3.4 mmol) in a50% aqueous solution of ethanol (24 mL) was stirred at room temperaturefor 12 h. The reaction mixture was concentrated in vacuo and theresulting residue was dissolved in ethyl acetate (12 mL). This reactionmixture was heated at reflux for 1 h and allowed to cool to roomtemperature. To this reaction mixture was added glacial acetic acid (0.2mL, 3.4 mmol). The separated aqueous layer was extracted with ethylacetate (4×10 mL). The combined organic extracts were washed with brine,dried (MgSO₄), filtered through Celite, and concentrated in vacuo toprovide 0.54 g of crude. Purification by radial PLC (silica gel, 25-50%ethyl acetate/hexanes) gave 0.35 g (63%) ofc-3-(tert-butoxycarbonylamino)-t-4-[(ethoxy-carbonyl)(methylcarbonylamino)methyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-olas a white solid, mp 58-59° C.

Analysis: Calculated for C₂₉H₃₈N₂O₆S₂: C, 60.60; H, 6.66; N, 4.87 Found:C, 60.76; H, 6.79; N, 4.88

To a stirred solution ofc-3-(tert-butoxycarbonylamino)-t-4-[(ethoxycarbonyl)-(methylcarbonylamino)methyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-ol(0.17 g, 0.29 mmol) in tetrahydrofuran (7 mL) at room temperature wasadded lithium borohydride (2 M, 0.3 mL, 0.6 mmol). After 12 h ofstirring, the reaction mixture was heated at 50° C. for 45 min. To thisreaction mixture was added 1 N HCl (6 mL). The separated aqueous layerwas extracted with ethyl acetate (4×5 mL). The combined organic extractswere washed with brine, dried (MgSO₄), filtered through Celite, andconcentrated in vacuo to provide 0.16 g of crude. Purification by radialPLC (silica gel, 50% ethyl acetate/hexanes) gave 0.08 g (50%) ofc-3-(tert-butoxycarbonyl-amino)-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-t-1-[bis(phenylthio)methyl]cyclo-pentan-r-1-ol(isomer A) as a white solid, mp 66-67° C.

Analysis: Calculated for C₂₇H₃₆N₂O₅S₂: C, 60.88; H, 6.62; N, 5.26 Found:C, 60.95; H, 6.94; N, 5.14

To a stirred solution ofc-3-(tert-butoxycarbonylamino)-t-4-[bis(ethoxycarbonyl)-(methylcarbonylamino)methyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-ol(0.76 g, 1.2 mmol) in tetrahydrofuran (10 mL) at room temperature wasadded lithium borohydride (0.03 g, 1.4 mmol). The reaction mixture washeated at reflux for 12 h and allowed to cool to room temperature. Tothis reaction mixture was added 1 N HCl (10 mL). The separated aqueouslayer was extracted with ether (4×10 mL). The combined organic extractswere washed with brine, dried (MgSO₄), filtered through Celite, andconcentrated in vacuo to provide 0.54 g of crude. Purification by radialPLC (silica gel, 70% ethyl acetate/hexanes) gave 0.15 g (21%) ofc-3-(tert-butoxycarbonylamino)-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-t-1-[bis-(phenylthio)-methyl]cyclopentan-r-1-ol(isomer B) as a white solid, mp 176-177° C.

Analysis: Calculated for C₂₇H₃₆N₂O₅S₂: C, 60.88; H, 6.62; N, 5.26 Found:C, 60.85; H, 6.72; N, 5.03

To a stirred solution of a mixture of isomers A and B ofc-3-(tert-butoxycarbonylamino)-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-ol(5.65 g, 10.6 mmol) in dimethylformamide (100 mL) at −23° C. was addedsodium hydroxide (95%, 0.345 g, 13.8 mmol) and tetrabutylammonium iodide(0.40 g, 1.1 mmol). After 30 min of stirring, benzyl bromide (2.0 mL,15.8 mmol) was added dropwise. The reaction mixture was stirred at −23°C. for 3 h then quenched with glacial acetic acid (2.5 mL) and water(100 mL). The separated aqueous layer was extracted with ethyl acetate(7×15 mL). The combined organic extracts were washed with brine, dried(MgSO₄), filtered through Celite, and concentrated in vacuo to provide9.1 g of crude. Purification by flash column chromatography (silica gel,210 g, 50-75% ethyl acetate/hexanes) gave 2.98 g (45%) ofc-3-(tert-butoxycarbonylamino)-t-4-[(1-methylcarbonyl-amino)(2-phenylmethoxy)ethyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-olas a white solid, mp 52-54° C.

Analysis: Calculated for C₃₄Hl₄₂N₂O₅S₂: C, 65.57; H, 6.79; N, 4.49Found: C, 65.52; H, 6.80; N, 4.45

A mixture ofc-3-(tert-butoxycarbonylamino)-t-4-[(1-methylcarbonylamino)(2-phenyl-methoxy)ethyl]-t-1-[bis-(phenylthio)methyl]cyclopentan-r-1-ol(2.53 g, 4.1 mmol), mercuric oxide (1.90 g, 8.8 mmol), and borontrifluoride etherate (1.1 mL, 8.9 mmol) in a 15% aqueous solution oftetrahydrofuran (70 mL) was stirred at room temperature for 2 h. Thereaction mixture was filtered through a pad of Celite and Florisil. Thefiltrate was concentrated in vacuo to give 2.74 g of the crude. To theabove crude in methanol (50 mL) was added iodine (1.9 g, 7.5 mmol) andthe reaction mixture was heated to 50° C. To this mixture was addeddropwise a solution of potassium hydroxide (0.71 M/methanol, 50 mL, 35.7mmol). After 2 h stirring at 50° C., the reaction mixture was filteredthrough Celite. The filtrate was concentrated in vacuo and the resultingresidue was dissolved in ethyl acetate (30 mL) and water (20 mL). Thelayers were separated and the aqueous layer was extracted with ethylacetate (4×10 mL). The combined organic extracts were washed with brine,dried (MgSO₄), filtered through Celite, and concentrated in vacuo toprovide 3.6 g of crude. Purification by flash column chromatography(silica gel, 200 g, 50-100% ethyl acetate/hexanes) gave 0.224 g (14%) ofthe desired hydroxyacid as a white solid. To a solution of hydroxyacid(0.244 g, 0.56 mmol) in dichloromethane (15 mL) was addedtrifluoroacetic acid (0.86 mL, 11.2 mmol) and the reaction mixture wasstirred at room temperature for 12 h. The reaction mixture wasconcentrated in vacuo to give crude. Purification by flash columnchromatography (silica gel, 60 g, chloroform/methanol/ammoniumhydroxide: 80/18/2) gave 0.241 g of a thick, yellow oil. Trituration ofthe yellow oil with ether provided 0.185 g (51%) oft-3-amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-hydroxycyclopentan-r-carboxylicacid as a tan solid, mp 37-39° C.

Analysis: Calculated for C₁₇H₂₄N₂O₅.C₂HF₃O.1.5C₂H₄F₃NO₂: C, 40.84; H,4.83; N, 7.58 Found: C, 40.86; H, 5.08; N, 7.90

A mixture oft-3-amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-hydroxycyclopentan-r-carboxylicacid (0.09 g, 0.14 mmol) and Palladium hydroxide (0.15 g) in ethanol (20mL) was hydrogenated at 40 psi overnight. The reaction mixture wasfiltered through Celite and the filtrate was concentrated in vacuo togive 0.078 g of crude. A mixture of the above crude amine andtrifluoroacetic acid (0.2 mL, 2.6 mmol) in dichloromethane (10 mL) wasstirred overnight. The mixture was concentrated in vacuo to give 0.08 gof a brown solid which was triturated with ether to provide 0.045 g(75%) of the title compound as a tan solid, mp 83-85° C.

Analysis: Calculated for C₁₀H₁₈N₂O₅.C₂HF₃O₂.0.5C₂H₄F₃NO₂: C, 36.67; H,4.97; N, 8.22 Found: C, 36.40; H, 5.15; N, 7.95

Example 9

t-3-{[(Amino)(imino)methyl]amino}-t-1-hydroxy-c-4-[(2-hydroxymethyl)(1-methylcarbonyl-amino)ethyl]cyclopentan-r-carboxylicacid trifluoroacetic acid diethyl ether [20:15:2] (isomer A at C-6)

To tris(methylthio)methane (21.27 g, 138 mmol) in tetrahydrofuran (350mL) at −78° C. was added dropwise over a period of 10 min under nitrogenn-butyllithium (1.6 M solution in hexane, 90 mL, 144 mmol) and stirredat −78° C. for 40 min. To the anion at −78° C. was added a solution of3β-(tert-butoxycarbonylamino)-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]-cyclopentanonefrom Example 6 (isomer A, 8.34 g, 17.23 mmol) in tetrahydrofuran (50 mL)over a period of 10 min and the reaction mixture stirred at −78° C. for1.5 h. The reaction was quenched with saturated ammonium chloride (50mL) and warmed to room temperature, ether (50 mL), added and the organiclayer was separated. The aqueous layer was extracted with ether (2×50mL), the organic layers were combined, dried over MgSO₄ and concentratedin vacuo to furnish an oil. The crude oil was dissolved in ethyl acetate(50 mL) and hexane (400 mL) and stored overnight in a freezer. Acrystalline solid was removed by filtration. The mother liquor waspurified by flash column chromatography (silica gel, 240 g, 30-45% ethylacetate in hexane) to obtain 2.7 g (24%) of3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]-1-[(tris-methythio)methyl]cyclopentanolas a mixture of isomers.

To a solution of above mixture (2.69 g, 4.22 mmol) in tetrahydrofuran(42 mL) was added dropwise under nitrogen lithium borohydride (0.74 g,33.73 mmol) and lithium 9-borabicyclo[3.3.1]nonane hydride (1 M solutionin tetrahydrofuran, 0.84 mL, 0.84 mmol) and the reaction mixture wasstirred at room temperature overnight. The reaction was quenched with 1N sodium hydroxide (1 mL), brine (20 mL) and stirred for 5 min. Thereaction was acidified to pH 4 using glacial acetic acid. Ether (10 mL)was added and the aqueous layer was separated. The aqueous layer wasneutralized with saturated aqueous sodium bicarbonate and extracted withethyl acetate (2×10 mL), the organic layers were combined, dried andconcentrated in vacuo to obtain 3.3 g of a yellow oil. The oil waspurified by flash column chromatography [silica gel (200 g), 10%chloroform:methanol:conc ammonium hydroxide (80:18:2) indichloromethane] to furnish 1.5 g (60%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol,a white solid [R_(f)=0.17, 20% chloroform:methanol:conc ammoniumhydroxide (80:18:2) in dichloromethane].

To a mixture of above solid (1.2 g, 2.0 mmol), mercuric chloride (2.02g, 7.45 mmol) and mercuric oxide (0.65 g, 3.02 mmol) was addedmethanol/water (46.2/3.8 mL) and the reaction mixture was stirred atroom temperature for 30 min. The mixture was filtered through a pad ofCelite and Florisil (20 g). The cake was washed with methanol (20 mL)and the filtrate concentrated in vacuo to furnish 2.1 g of whitesemisolid. The crude was purified by flash column chromatography [silicagel (60 g); 75% ethyl acetate in hexane and 10% methanol in ethylacetate] to furnish 0.55 g (55%) of methylt-4-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-3-[(2-hydroxymethyl)(1-methylcarbonylamino)ethyl]-t-1-hydroxycyclopentan-r-carboxylateas a white solid, mp 94-96° C.

To a solution of above solid (0.47 g, 0.93 mmol) in tetrahydrofuran (9.3mL) was added 1 N sodium hydroxide (1.86 mL, 1.86 mmol) and water (7.4mL). The reaction mixture was stirred at room temperature for 1 h.Tetrahydrofuran was removed in vacuo and the aqueous layer was washedwith ether (2×10 mL). The aqueous layer was made acidic with glacialacetic acid (pH=5), saturated with sodium chloride and extracted withethyl acetate (3×15 mL). The organic layers were combined andconcentrated in vacuo to furnish 0.33 g (73%) of crude which wastriturated with ether/hexane to furnisht-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-c-4-[(2-hydroxymethyl)(1-methylcarbonylamino)ethyl]cyclo-pentan-r-carboxylicacid (isomer A) as a white solid, mp 238-240° C.

Analysis: Calculated for C₂₁H₃₆N₄O₉: C, 51.63; H, 7.43; N, 11.47 Found:C, 51.31; H, 7.48; N, 11.07

To a solution oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)-methyl]amino}-t-1-hydroxy-c-4-[(hydroxymethyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid, isomer A (0.2 g, 0.41 mmol), in dichloromethane (10 mL),trifluoroacetic acid (0.63 mL, 8.2 mmol) was added and the reaction wasstirred at room temperature overnight. Additional trifluoroacetic acid(0.32 mL, 4.1 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces ofexcess trifluoroacetic acid were removed in vacuo by co-distilling theresidue twice with dichloromethane (10 mL). The residue

Example 10

3β-amino-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentancarboxylicacid trifluoroacetic acid (1:1) (isomer A at C-6)

To 2-trimethylsilyl-1,3-dithiane (Aldrich, 7.88 g, 41.5 mmol) intetrahydrofuran (100 mL) at 0° C. was added dropwise over a period of 10min under nitrogen n-butyllithium (1.6 M solution in hexane, 28.6 mL,45.7 mmol) and stirred at 0° C. for 45 min. The anion was cooled to −40°C. and a solution of3β-(tert-butoxycarbonylamino)-4α-[bis(ethoxycarbonyl)(methylcarbonyl-amino)methyl]cyclopentanone(from Example 8, 4.3 g, 10.4 mmol) in tetrahydrofuran (50 mL) was thenadded dropwise over a period of 15 min. The reaction mixture was stirredat −40° C. for 5 h and warmed to −20° C. The reaction was quenched withsaturated ammonium chloride (50 mL) and warmed to room temperature.Ether (20 mL) was added and the organic layer was separated. The aqueouslayer was extracted with ether (2×25 mL), the organic layers werecombined, dried over MgSO₄ was dissolved in water (5 mL) andconcentrated in vacuo to furnish an oily residue which was trituratedwith ether to obtain 0.13 g (83%) of the title compound as a whitesolid, mp 162-166° C.

Analysis: Calculated for C₁₃H₁₉F₃N₄O₉.0.75C₂HF₃O₂.0.1C₄H₁₀O: C, 40.64;H, 5.75; N, 14.70 Found: C, 40.77; H, 5.80; N, 14.67

and concentrated in vacuo to furnish crude. Purification of the crude byflash column chromatography (silica gel, 320 g, 30-35% ethyl acetate inhexane) gave 3.16 g (59%) of2-{3β-(tert-butoxycarbonylamino)-4α-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentyl-idine}-1,3-dithianeas a colorless oil that solidified on drying in vacuo at acetone refluxtemperature to give a solid, mp 66-68° C.

Analysis: Calculated for C₂₃H₃₆N₂O₇S₂: C, 53.47; H, 7.02; N, 5.42 Found:C, 53.50; H, 7.07; N, 5.41

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[bis(ethoxycarbonyl)(methyl-carbonylamino)methyl]cyclopentylidene}-1,3-dithiane(7.5 g, 14.53 mmol) in ethanol (75 mL) was added 1 N sodium hydroxide(50.9 mL, 50.9 mmol) and water (25 mL) and the reaction mixture washeated at reflux for 2 h. The reaction was cooled, glacial acetic acid(4.6 mL, 76.3 mmol) was added, the mixture heated at gentle reflux for 1h and stirred at room temperature overnight. The solid that separatedwas collected by filtration, washed with water and dried in vacuo attoluene reflux temperature to furnish 1.63 g (27%) of solid. Thefiltrate was extracted with ethyl acetate (3×100 mL), the organic layerscombined, dried and concentrated in vacuo to furnish 3.5 g of residue.An analytical sample was prepared by crystallization of the combinedsolid from ethanol to furnish 5.1 g (85%) of2-{3β-(tert-butoxycarbonylamino)-4α-[(carboxy)(methylcarbonyl-amino)methyl]cyclopenylidiene}-1,3-dithianeas a white solid mp 174-176° C.

Analysis: Calculated for C₁₈H₂₈N₂O₅S₂.0.75H₂O: C, 50.27; H, 6.91; N,6.51 Found: C, 50.03; H, 6.54; N, 6.41

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(carboxy)(methylcarbonyl-amino)methyl]cyclopentylidene}-1,3-dithiane(5.13 g, 12.3 mmol) in tetrahydrofuran (120 mL) cooled to 0° C. wasadded methyl chloroformate (1 mL, 13.5 mmol) and triethylamine (2.2 mL,15.4 mmol). The reaction mixture was stirred at 0° C. for 40 min and acold solution of N,O-dimethylhydroxylamine hydrochloride (1.84 g, 18.5mmol) and triethylamine (3.5 mL, 24.6 mmol) in tetrahydrofuran (5 mL)that had been stirred at 0° C. for 30 min was added. The reactionmixture was allowed to warm to room temperature and stirred overnight.The reaction was filtered through Celite and the cake washed withtetrahydrofuran (10 mL). To the filtrate was added a cold solution ofN,O-dimethylhydroxylamine hydrochloride (1.84 g, 18.5 mmol) andtriethylamine (3.5 mL, 24.66 mmol) in tetrahydrofuran (5 mL) that hadbeen stirred at 0° C. for 30 min and again stirred overnight at roomtemperature. The solvent was removed in vacuo and to the residue sodiumhydroxide (0.1M, 100 mL) and ethyl acetate (100 mL) were added. Theorganic layer was separated and the aqueous layer was extracted withethyl acetate (2×75 mL). The organic layers were combined and washedwith brine (100 mL), dried and concentrated in vacuo to furnish 4.73 gcrude amide as a semisolid. Purification of the crude by flash columnchromatography [200 g silica gel, 90% ethyl acetate in hexane and 25%chloroform/methanol/ammonium hydroxide (80:18:2) in methylene chloride]gave 4.2 g (74%) of2-{3β-(tert-butoxycarbonylamino)-4α-{(methylcarbonylamino){[(methyl)(methoxy)amino]carbonyl}methyl}cyclopentylidene}-1,3-dithiane.An analytical sample, mp 122-126° C., was prepared as a white solid byrecrystallization from ether/hexane.

Analysis: Calculated for C₂₀H₃₃N₃O₅S₂: C, 52.26; H, 7.24; N, 9.14 Found:C, 52.34; H, 7.20; N, 9.09

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-{(methylcarbonylamino)-{[(methyl)(methoxy)amino]carbonyl}methyl}cyclopentylidene}-1,3-dithiane(0.26 g, 0.57 mmol) in tetrahydrofuran (2.5 mL) cooled to 0° C. wasadded dropwise lithium tri-tert-butoxyaluminohydride (Aldrich, 1.0 Msolution in tetrahydrofuran, 1.4 mL, 1.4 mmol). The reaction mixture wasstirred at room temperature overnight. The reaction was quenchedcarefully with 1 N HCl (1.0 mL, pH should not go below 4.0) and stirredfor 5 min. Ether (20 mL) and 1.0 M aqueous solution of sodium potassiumtartrate salt (10 mL) was added and reaction mixture was stirred at roomtemperature for 30 min. The organic layers were separated and aqueouslayer was extracted with ether (2×10 mL). The organic layers werecombined, washed with brine (20 mL) dried and concentrated in vacuo tofurnish 0.3 g of a white solid. Purification of the crude by flashcolumn chromatography (20 g silica gel, 50-80% ethyl acetate in hexane)gave two isomers (at C-6) of2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)(methylcarbonylamino)methyl]cyclopentylidene}-1,3-dithiane:

1. 0.09 g (40%) of isomer A at C-6, a white solid, mp 188-192°0 C.(dec).

Analysis: Calculated for C₁₈H₂₈N₂O₄S₂: C, 53.97; H, 7.05; N, 6.99 Found:C, 53.93; H, 7.09; N, 6.93

2. 0.08 g (35%) of isomer B at C-6, a white solid, mp >180° C. (dec).

Analysis: Calculated for C₁₈H₂₈N₂O₄S₂: C, 53.97; H, 7.05; N, 6.99 Found:C, 54.03; H, 7.05; N, 6.97

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)(methylcarbonyl-amino)methyl]cyclopentylidene}-1,3-dithiane(0.17 g, 0.43 mmol) in tetrahydrofuran (5 mL) cooled to −78° C. wasadded dropwise vinylmagnesium bromide (Aldrich, 1.0 M solution intetrahydrofuran, 2.2 mL, 2.2 mmol) and stirred at −78° C. for 2 h. Thereaction was quenched carefully with saturated aqueous ammonium chloride(5.0 mL). Ether (10 mL) and brine (5 mL) were added and the reactionmixture was allowed to warm to room temperature. The organic layers wereseparated and the aqueous layer was extracted with ether (2×10 mL). Theorganic layers were combined, washed with brine (20 mL) dried andconcentrated in vacuo to furnish 0.17 g crude as a white solid.Purification of the crude by flash column chromatography (10 g silicagel, 50-100% ethyl acetate in hexane) gave 0.06 g (33%) of2-{3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentylidene}-1,3-dithiane(isomer A at C-6) as a white solid, mp >210° C. (dec).

Analysis: Calculated for C₂₀H₃₂N₂O₄S₂: C, 56.05; H, 7.53; N, 6.54 Found:C, 56.18; H, 7.50; N, 6.47

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonyl-amino)-3-butenyl]cyclopentylidene}-1,3-dithiane(isomer A, 0.55 g, 1.28 mmol) in methanol (19.3 mL) was added 6 N HCl(3.2 mL, 19.28 mmol) and the mixture was stirred at room temperatureuntil all starting material had disappeared (TLC, ethyl acetate and MSanalysis ˜20 h). The reaction mixture was cooled to 0° C. and sodiumhydroxide (1.02 g, 25.7 mmol) was added and the reaction was stirred atroom temperature for 1 h. The reaction was quenched with glacial aceticacid (0.8 mL, 12.85) and concentrated in vacuo to furnish crude residue.To the residue was added ethyl acetate (10 mL) and water (10 mL). Theaqueous layer was separated and extracted with ethyl acetate (2×10 mL).The organic layers were combined and concentrated in vacuo to furnish0.4 g of crude.

The above crude was dissolved in anhydrous methanol (20 mL) and cooledto 0° C. A solution of dry HCl in ether (Aldrich, 1.0 M solution, 5 mL)was added and the reaction mixture was stirred overnight. The reactionwas quenched with 1 N sodium hydroxide in methanol to adjust the pH ofthe reaction to 6-7, and concentrated in vacuo to obtain crude residue.The residue was dissolved in water (10 mL) and extracted withdichloromethane (3×10 mL). The organic layers were combined, dried andconcentrated in vacuo to obtain 0.2 g of crude. The crude was purifiedby flash column chromatography [10-50% chloroform:methanol:conc ammoniumhydroxide (80:18:2) in dichloromethane] to furnish 0.12 g (24%) ofmethyl3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentan-carboxylate(isomer A at C-6) as an oil, MS (ES+) 371.4 [100%, (M+1)] and 353.4[100%, (M+1)−H₂O].

The above oil (0.1 g, 0.27 mmol) was dissolved intetrahydrofuran/tert-butanol (2 mL, 1:1) and N-methyl morpholine oxide(50 mg), osmium tetraoxide (0.05 wt % in tert-butanol, 0.2 mL) and water(1 mL) was added. The reaction mixture was stirred overnight at roomtemperature. A saturated aqueous solution of sodium sulfite (2 mL) wasadded and stirred vigorously for 30 min. Brine (2 mL) was added theaqueous layer was extracted with ethyl acetate (3×10 mL). The organiclayers were combined, dried and concentrated in vacuo to obtain 0.1 g ofcrude. The crude was purified by flashcolumn chromatography [0, 5, 10,50, 100% methanol in ethyl acetate] to furnish 0.06 g (59%) of triol asa semisolid, MS (ES+) 405.4 [100%, (M+1)] and 387.5 [60%, (M+1)−H₂O]. Toa solution of the above solid (0.06 g, 0.16 mmol) in tetrahydrofuran(1.6 mL) was added 1 N sodium hydroxide (1.0 mL, 1.0 mmol) and stirredat room temperature for 1.5 h. Ether (5 mL) and water (1 mL) were added,and organic layers were separated. The aqueous layer was washed withethyl acetate (2×5 mL). The aqueous layer was then acidified to pH 5-4using 1 N HCl, saturated with sodium chloride and extracted with ethylacetate (3×5 mL. The organic layers were combined, dried andconcentrated in vacuo to furnish 0.027 g (43%) of3β-(tert-butoxycarbonylamino)-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentancarboxylicacid (isomer A at C-6) as a white solid, MS (ES+) 391.4 [55%, (M+1)],373.6 [40% (M+1)−H₂O] and 100% 317.3 [(M+1)-tert-butyl].

To a solution of above acid (0.027 g, 0.07 mmol) in dichloromethane (1.0mL) was added trifluoroacetic acid (0.11 mL, 1.4 mmol), and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.11 mL, 1.4 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces oftrifluoroacetic acid were removed in vacuo by co-distilling the residuetwice with dichloromethane (10 mL). The residue was dissolved in water(0.5 mL), concentrated in vacuo and dried at acetone reflux temperaturein vacuo to obtain 0.017 g (60%) of the title compound (isomer A) as atan solid, MS (ES+) 291.4 [100%, (M+1)].

Example 11

3β-amino-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclooentancarboxylicacid trifluoroacetic acid (1:2) (isomer B at C-6)

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)(methylcarbonyl-amino)methyl]cyclopentylidene}-1,3-dithiane(isomer B at C-6), from Example 10 (0.45 g, 1.13 mmol) intetrahydrofuran (20 mL) cooled to −78° C. was added dropwisevinylmagnesium bromide (Aldrich, 1.0 M solution in tetrahydrofuran, 5.6mL, 5.6 mmol) and stirred at −78° C. for 1 h. The reaction was quenchedcarefully with saturated aqueous ammonium chloride (5.0 mL). Ether (20mL) and brine (5 mL) were added and the reaction mixture was allowed towarm to room temperature. The organic layers were separated and theaqueous layer was extracted with ether (2×10 mL). The organic layerswere combined, washed with brine (20 mL), dried and concentrated invacuo to furnish 0.5 g of a white solid. Purification by flash columnchromatography (10 g silica gel, 60-100% ethyl acetate in hexane) gave0.21 g (44%) of2-{3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentylidene}-1,3-dithiane(isomer B at C-6) as a white solid, mp >210° C. (dec).

Analysis: Calculated for C₂₀H₃₂N₂O₄S₂.0.25H₂O: C, 55.46; H, 7.56; N,6.47 Found: C, 55.20; H, 7.47; N, 6.41

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonyl-amino)-3-butenyl]cyclopentylidene}-1,3-dithiane(isomer B at C-6) (0.58 g, 1.36 mmol) in methanol (21 mL) was added 6 NHCl (3.4 mL, 20.46 mmol) and the mixture was stirred at room temperatureuntil all starting material had disappeared (˜20 h). The reactionmixture was cooled to 0° C. and sodium hydroxide (1.1 g, 27.6 mmol) wasadded and the reaction was stirred at room temperature for 1 h. Thereaction was quenched with glacial acetic acid (0.83 mL, 13.79 mmol) andconcentrated in vacuo. To the residue obtained was added ethyl acetate(10 mL) and water (10 mL). The aqueous layer was separated and extractedwith ethyl acetate (2×10 mL). The organic layers were combined andconcentrated in vacuo to furnish 0.41 g of residue.

The above crude was dissolved in anhydrous methanol (20 mL) and cooledto 0° C. A solution of dry HCl in ether (Aldrich, 1.0M solution, 5 mL)was added and the reaction mixture was stirred overnight. The reactionwas quenched with 1 N sodium hydroxide in methanol to adjust pH of thereaction to 6-7 and then concentrated in vacuo. The residue wasdissolved in water (10 mL) and extracted with dichloromethane (3×10 mL).The organic layers were combined, dried and concentrated in vacuo toobtain 0.17 g of crude. The crude was purified by flash columnchromatography [10-50% chloroform:methanol:conc ammonium hydroxide(80:18:2) in dichloromethane] to furnish 0.14 g (28%) of methyl3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylate(isomer B at C-6) as an oil, MS (ES+) 371.4 [90%, (M+1)].

The above oil (0.12 g, 0.32 mmol) was dissolved intetrahydrofuran/tert-butanol (2 mL, 1:1) and N-methylmorpholine oxide(50 mg), osmium tetraoxide (0.05 wt % in tert-butanol, 0.2 mL) and water(1 mL) was added. The reaction mixture was stirred overnight at roomtemperature. A saturated aqueous solution of sodium sulfite (2 mL) wasadded and stirred vigorously for 30 min. Brine (2 mL) was added and theaqueous layer was extracted with ethyl acetate (3×10 mL). The organiclayers were combined, dried and concentrated in vacuo to obtain 0.1 g ofcrude. The crude was purified by flash column chromatography [0, 5, 10,50, 100% methanol in ethyl acetate] to furnish 0.08 g (62%) of the triolas a semisolid, MS (ES+) 405.2 [100%, (M+1)].

To a solution of above solid (0.08 g, 0.2 mmol) in tetrahydrofuran (2mL) was added 1 N sodium hydroxide (1.2 mL, 1.2 mmol) and stirred atroom temperature for 1.5 h. Ether (5 mL) and water (2 mL) were added ,and the organic layers were separated. The aqueous layer was washed withethyl acetate (2×5 mL). The aqueous layer was acidified to pH 5-4 using1 N HCl saturated with sodium chloride, and extracted with ethyl acetate(3×5 mL). The organic layers were combined, dried and concentrated invacuo to furnish 0.02 g (26%) of3β-(tert-butoxycarbonylamino)-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentan-carboxylicacid (isomer B at C-6) as a white solid, MS (ES+) 391.4 [20%, (M+1)] and373.6 [100%, (M+1)−H₂O].

To a solution of above acid (0.02 g, 0.05 mmol) in dichloromethane (1.0mL) was added trifluoroacetic acid (0.08 mL, 1.1 mmol) and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.08 mL, 1.1 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces oftrifluoroacetic acid were removed in vacuo by co-distilling the residuetwice with dichloromethane (10 mL). The residue was dissolved in water(0.5 mL), concentrated in vacuo and dried at acetone reflux temperaturein vacuo to obtain 0.2 g (77%) of the title compound as a tan solid, mp58-62° C.

Analysis: Calculated for C₁₂H₂₂N₂O₆.2C₂HF₃O₂: C, 37.08; H, 4.67; N, 5.40Found: C, 37.50; H, 4.43; N, 5.28

Example 12

3β-{[(Amino)(imino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl)butyl]-cyclonentancarboxylic acidtrifluoroacetic acid (1:2) (isomer A at C-1, isomer A at C-6, isomer Bat C-7)

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)methylcarbonylamino)-methyl]cyclopentylidene}-1,3-dithane(isomer A at C-6), from Example 10 (1.75 g, 4.4 mmol), intetrahydrofuran (100 mL) cooled to −78° C. was added dropwisevinylmagnesium bromide (Aldrich, 1.0 M solution on tetrahydrofuran, 44mL, 44 mmol) and stirred at −78° C. for 2 h. The reaction was quenchedcarefully with saturate aqueous ammonium chloride (20 mL), ether (100mL) and brine (20 mL) was added and the reaction mixture was allowed towarm to room temperature. The organic layers were separate and theaqueous layer was extracted with ether (3×50 mL) and dichloromethane(100 mL). The organic layers were combined, dried and concentrated invacuo to furnish 2.0 g of a white solid.

To the above solid (2.0 g, 4.4 mmol) dissolved in dimethylformamide (20mL) was added tert-butyldimethylsilyl chloride (0.86 g, 5.5 mmol),imidazole (0.6 g, 8.8 mmol) and 4-dimethylaminopyridine (0.14 g, 0.11mmol). The reaction mixture was stirred at room temperature overnight.The reaction was quenched with water (20 mL), and extracted with ether(3×25 mL), dried and concentrated in vacuo to furnish 2.48 g of crude.Purification of the crude by flash column chromatography (150 g silicagel, 10-30% ethyl acetate in hexane) gave the following isomers of2-{3β-(tert-butoxycarbonylamino)-4α-{2-{[(tert-butyl)(dimethyl)silyl]oxy}-3-butenyl}cyclopentylidene}-1,3-dithiane:

1. 0.22 g (9%) of isomer A at C-6, isomer A at C-7 as a white solid, mp72-76° C. (dec).

Analysis: Calculated for C₂₆H₄₆N₂O₄S₂Si.0.5H₂O: C, 56.59; H, 8.59; N,5.08 Found: C, 56.61; H, 8.43; N, 4.97

2. 0.86 g (36%) of isomer A at C-6, isomer B at C-7 as a white solid, mp116-118° C.

Analysis Calculated for C₂₆H₄₆N₂O₄S₂Si: C, 57.53; H, 8.54; N, 5.16Found: C, 57.84; H, 8.59; N, 5.23

To a solution of isomer A at C-6, isomer B at C-7 from above (0.58 g,1.07 mmol) in methanol (16.6 mL) was added 6 N HCl (2.8 mL, 16.6 mmol)and the mixture was stirred at room temperature overnight. The reactionmixture was cooled to 0° C. and sodium hydroxide (0.86 g, 10.7 mmol) wasadded and the reaction was stirred at room temperature for 1 h. Thereaction was quenched with glacial acetic acid (0.64 mL, 10.7 mmol) andconcentrated in vacuo. To the residue obtained was added 1 N HCl (2.14mL, 2.14 mmol), ethyl acetate (10 mL) and water (10 mL). The aqueouslayer was separated, saturated with sodium chloride and extracted withethyl acetate (2×10 mL). The organic layers were combined andconcentrated in vacuo to furnish 0.33 g (85%) of crude.

The above crude was dissolved in anhydrous methanol (8 mL) and cooled to0° C. A solution of dry HCl in ether (Aldrich, 1.0 M solution, 1.6 mL)was added and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was concentrated in vacuo (bathtemperature 25° C.) to obtain a crude residue of methyl3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylateas an oil, MS (ES+) 371.5 [100%, (M+1)].

To a solution of above compound in dichloromethane (8.0 mL) was added(1.26 mL, 16.4 mmol) trifluoroacetic acid, and the reaction was stirredat room temperature overnight. Additional trifluoroacetic acid (0.63 mL,8,2 mmol) was added and the reaction was stirred at room temperature for1 h. The solvent was removed in vacuo and traces of trifluoroacetic acidwere removed in vacuo by co-distilling the residue twice withdichloromethane (5 mL). The residue was dissolved in dimethylformamide(5 mL), triethylamine (0.58 mL, 4.1 mmol),1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (0.29 g, 0.98mmol), and mercuric chloride (0.27 g, 0.98 mmol) were added, and thereaction was stirred overnight at room temperature. The reaction mixturewas diluted with ethyl acetate (20 mL) and filtered to remove inorganicimpurities. The filtrate was washed with water (2×10 mL) and brine (10mL), dried and concentrated in vacuo to obtain 0.4 g of crude. The crudewas purified by flash column chromatography [silica gel (20 g), 40-50%ethyl acetate in hexane] to furnish the two C-1 isomers of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylate:

1. 0.15 g (27%) of isomer A at C-1, as a semisolid, MS (ES+) 495.5[100%, (M+1)−H₂O] and 513.6 [10%, (M+1)].

2. 0.07 g (13%) of isomer B at C-1, as a semisolid, MS (ES+) 513.5[100%, (M+1)].

The above isomer A (0.15 g, 0.29 mmol) was dissolved intetrahydrofuran/tert-butanol (2 mL, 1:1) and N-methyl morpholine oxide(50 mg), osmium tetraoxide (few crystals) and water (1 mL) were added.The reaction mixture was stirred overnight at room temperature. Asaturated, aqueous solution of sodium sulfite (2 mL) and sodium sulfite(1 g) was added and stirred vigorously for 30 min. Brine (2 mL) wasadded and the aqueous layer was saturated with sodium chloride andextracted with ethyl acetate (3×10 mL). The organic layers werecombined, dried and concentrated in vacuo to obtain 0.15 g (94%) of puremethyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(1-methylcarbonylamino)-(2,3,4-trihydroxy)butyl]cyclopentancarboxylate(isomer A at C-1), MS (ES+) 529.4 [100%, (M+1)−H₂O] and 547.4 [50%,(M+1)].

To a solution of above solid (0.15 g, 0.27 mmol) in tetrahydrofuran (1.5mL) was added 1 N sodium hydroxide (1.4 mL, 1.4 mmol), and stirred atroom temperature for 2.0 h. Ether (2 mL) and water (2 mL) were added andorganic layers were separated. The aqueous layer was washed with ether(2×5 mL). The aqueous layer was acidified to pH 4 using 1 N HClsaturated with sodium chloride and extracted with ethyl acetate (3×5mL). The organic layers were combined, dried and concentrated in vacuoto furnish 0.13 g (84%) of acid as a white solid, MS (ES+) 515.4 [80%,(M+1)−H₂O] and 533.5 [30%, (M+1)]

To a solution of the above acid (0.13 g, 0.24 mmol) in dichloromethane(2.0 mL) was added trifluoroacetic acid (0.04 mL, 0.48 mmol) and thereaction was stirred at room temperature overnight. Additionaltrifluoroacetic acid (0.02 mL, 0.24 mmol) was added and the reaction wasstirred at room temperature for 1 h. The solvent was removed in vacuoand traces of excess trifluoroacetic acid were removed in vacuo byco-distilling the residue twice with dichloromethane (10 mL). Theresidue was dissolved in water (1.0 mL), concentrated in vacuo and driedat acetone reflux temperature in vacuo to obtain 0.07 g (46%) of thetitle compound as a tan solid, mp 76-80° C.

Analysis: Calculated for C₁₃H₂₄N₄O₆.2C₂HF₃O₂: C, 36.44; H, 4.67; N, 9.99Found: C, 36.81; H, 4.24; N, 9.55

Example 13

3β-{[(Amino)(imino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]-cyclopentancarboxylicacid trifluoroacetic acid (1:2) (isomer B at C-1, isomer A at C-6,isomer B at C-7)

Methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylate(isomer B at C-1, from Example 12) (70 mg, 0.14 mmol), was dissolved intetrahydrofuran/tert-butanol (2 mL, 1:1) and N-methyl morpholine oxide(50 mg), osmium tetraoxide (few crystals) and water (1 mL) were added.The reaction mixture was stirred overnight at room temperature. Asaturated aqueous solution of sodium sulfite (2 mL) and sodium sulfite(1 g) was added and stirred vigorously for 30 min. Brine (2 mL) wasadded and the aqueous layer was saturated with sodium chloride andextracted with ethyl acetate (3×10 mL). The organic layers werecombined, dried and concentrated in vacuo to obtain 0.07 g (86%) ofmethyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclo-pentancarboxylate(isomer B at C-1), MS (ES+) 547.4 [100%, (M+1)].

To a solution of above solid (0.07 g, 0.13 mmol) in tetrahydrofuran (1.5mL) was added 1 N sodium hydroxide (0.64 mL, 0.64 mmol) and stirred atroom temperature for 2 h. Ether (2 mL) and water (2 mL) were added andorganic layers were separated. The aqueous layer was washed with ether(2×5 mL). The aqueous layer was acidified to pH 4 using 1 N HCl,saturated with sodium chloride, and extracted with ethyl acetate (3×5mL). The organic layers were combined, dried and concentrated in vacuoto furnish 0.017 g (26%) of acid as a white solid, MS (ES+) 533.2 [60%,(M+1)].

To a solution of above acid (0.017 g, 0.03 mmol) in dichloromethane (2.0mL) was added trifluoroacetic acid (0.06 mL, 0.6 mmol) and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.03 mL, 0.3 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces ofexcess trifluoroacetic acid were removed in vacuo by co-distilling theresidue twice with dichloromethane (10 mL). The residue was dissolved inwater (1.0 mL) concentrated in vacuo and dried at acetone refluxtemperature in vacuo to obtain 0.01 g (60%) of the title compound as atan solid, mp 124-128° C.

Analysis: Calculated for C₁₃H₂₄N₄O₆.2C₂HF₃O₂: C, 36.44; H, 4.67; N,10.00 Found: C, 35.84; H, 4.24; N, 10.51

Example 14

3β-{[(Amino)(imino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclo-pentancarboxylicacid trifluoroacetic acid (4:9) (isomer B at C-6, isomer A at C-1, C-7and/or C-8)

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)(methylcarbonylamino)-methyl]cyclopentylidene}-1,3-dithiane(isomer B at C-6) (2.58 g, 6.45 mmol) from Example 10 in tetrahydrofuran(64 mL) cooled to −78° C. was added dropwise vinylmagnesium bromide(Aldrich, 1.0 M solution in tetrahydrofuran, 64.5 mL, 64.5 mmol) and themixture stirred at −78° C. for 1 h. The reaction was quenched carefullywith saturated aqueous ammonium chloride (20 mL). Ether (100 mL) andbrine (20 mL) were added and reaction mixture was allowed to warm toroom temperature. The organic layers were separated and the aqueouslayer was extracted with ether (3×50 mL). The organic layers werecombined, dried and concentrated in vacuo to furnish 3.7 g of a whitesolid.

To the above solid (2.0 g, 4.4 mmol) dissolved in dimethylformamide (30mL) was added tert-butyldimethylsilyl chloride (1.31 g, 8.44 mmol),imidazole (0.92 g, 13.5 mmol) and dimethylaminopyridine (0.21 g, 1.69mmol). The reaction mixture was stirred at room temperature overnight.The reaction was quenched with water (30 mL) and extracted with ether(3×30 mL). The organic layers were combined and washed with water (30mL) and brine (30 mL), dried and concentrated in vacuo to furnish 3.8 gof crude. Purification of the crude by flash column chromatography (210g silica gel, 25% ethyl acetate in hexane) gave 1.44 g (41%) of2-{3β-(tert-butoxycarbonylamino)-4α-[(1-methylcarbonylamino)(2-{[(tert-butyl)(dimethyl)silyl]oxy}-3-butenyl]cyclopentylidene}-1,3-dithiane(isomer B at C-6, 85:15 mixture of isomers at C-7) as a white solid, mp76-84° C.

Analysis: Calculated for C₂₆H₄₆N₂O₄S₂Si: C, 57.53; H, 8.54; N, 5.16Found: C, 57.29; H, 8.52; N, 5.09

To a solution of2-{3β-(tert-butoxycarbonylamino)-4α-[(1-methylcarbonylamino)(2-{[(tert-butyl)(dimethyl)silyl]oxy}-3-butenyl]cyclopentylidene}-1,3-dithiane(1.4 g, 2.58 mmol) in methanol (39.0 mL) was added 6 N HCl (6.5 mL, 39.0mmol) and the mixture was stirred at room temperature overnight. Thereaction mixture was cooled to 0° C. and sodium hydroxide (2.07 g, 51.7mmol) was added and the reaction was stirred at room temperature for 1h. The reaction was quenched with glacial acetic acid (1.6 mL, 27 mmol)and concentrated in vacuo. To the residue obtained was added 1 N HCl(5.2 mL, 5.2 mmol), ethyl acetate (10 mL) and water (20 mL). The aqueouslayer was separated, saturated with sodium chloride and extracted withethyl acetate (2×10 mL). The organic layers were combined andconcentrated in vacuo to furnish 0.68 g (74%) of crude.

The above crude was dissolved in anhydrous methanol (19 mL) and cooledto 0° C. A solution of dry HCl in ether (Aldrich, 1.0 M solution, 3.8mL) was added and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was concentrated in vacuo (bathtemperature 25° C.) to obtain methyl3β-(tert-butoxycarbonylamino)-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylateas an oil, MS (ES+) 371.5 [100%, (M+1)].

To a solution of above ester in dichloromethane (19 mL) was added (2.94mL, 38.0 mmol) of trifluoroacetic acid and the reaction was stirred atroom temperature overnight. Additional trifluoroacetic acid (1.5 mL, 17mmol) was added and the reaction was stirred at room temperature for 1h. The solvent was removed in vacuo and traces of trifluoroacetic acidwere removed in vacuo by co-distilling the residue twice withdichloromethane (5 mL). The residue was dissolved in dimethylformamide(10 mL) and triethylamine (1.4 mL, 10 mmol),1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (0.66 g, 2.28mmol), and mercuric chloride (0.62 g, 2.28 mmol) were added. Thereaction was stirred overnight at room temperature, and the reactionmixture was diluted with ethyl acetate (30 mL) and filtered to removeinorganic impurities. The filtrate was washed with water (2×10 mL) andbrine (10 mL), dried and concentrated in vacuo to obtain 0.78 g ofcrude. The crude was purified by flash column chromatography [silica gel(40 g), 40-50% ethyl acetate in hexane] to furnish two isomers of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methyl-carbonylamino)-3-butenyl]cyclopentancarboxylate:

1. 0.15 g (16%) of isomer A as a semisolid, MS (ES+) 495.5 [100%(M+1)−H₂O] and 513.6 [30%, (M+1)].

2. 0.12 g (13%) of isomer B as a semisolid, MS (ES+) 513.5 [100%,(M+1)].

The above isomer A (0.13 g, 0.25 mmol) was dissolved intetrahydrofuran/tert-butanol (2 mL, 1:1) and N-methylmorpholine oxide(50 mg), osmium tetraoxide (few crystals) and water (1 mL) were added.The reaction mixture was stirred overnight at room temperature. Asaturated aqueous solution of sodium sulfite (2 mL) and sodium sulfite(1 g) was added and stirred vigorously for 30 min. Brine (2 mL) wasadded and the aqueous layer was saturated with sodium chloride andextracted with ethyl acetate (3×10 mL). The organic layers werecombined, dried and concentrated in vacuo to obtain 0.12 g (92%) ofmethyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]-cyclopentancarboxylate,MS (ES+) 547.4 [20%, M+1)] and 529.4 [100%, (M+1)−H₂O].

To a solution of above solid (0.12 g, 0.23 mmol) in tetrahydrofuran (1mL) was added 1 N sodium hydroxide (1.1 mL, 1.1 mmol) and the mixturestirred at room temperature for 2.0 h. Ether (2 mL) and water (2 mL)were added, the layers were separated and the aqueous layer wassaturated with sodium chloride and extracted with ethyl acetate (3×5mL). The organic layers were combined, dried and concentrated in vacuoto furnish 0.033 g (25%) of acid as a white solid, MS (ES+) 515.4 [25%,(M+1)−H₂O] and 533.4 [5%, (M+1)].

To a solution of above acid (0.033 g, 0.06 mmol) in dichloromethane (2.0mL) was added trifluoroacetic acid (0.1 mL, 1.2 mmol) and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.05 mL, 0.6 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces ofexcess trifluoroacetic acid were removed in vacuo by co-distilling theresidue twice with dichloromethane (10 mL). The residue was dissolved inwater (1.0 mL), concentrated in vacuo and dried at acetone refluxtemperature in vacuo to obtain 0.018 g (49%) of the title compound as atan solid, mp 125-135° C.

Analysis: Calculated for C₁₃H₂₄N₄O₆.2.25C₂HF₃O₂: C, 35.69; H, 4.49; N,9.51 Found: C, 36.02; H, 4.19; N, 9.58

Example 15

3β-{[(Amino)(imino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclo-pentancarboxylicacid trifluoroacetic acid (5:13) (isomer B at C-6, isomer B at C-1, C-7and/or C-8)

Methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)-3-butenyl]cyclopentancarboxylate(isomer B at C-6, isomer B at C-1 and C-7, from Example 14) (0.12 g,0.24 mmol) was dissolved in tetrahydrofuran/tert-butanol (2 mL, 1:1) andN-methylmorpholine oxide (60 mg), osmium tetraoxide (few crystals) andwater (1 mL) was added. The reaction mixture was stirred overnight atroom temperature. A saturated aqueous solution of sodium sulfite (2 mL)and sodium sulfite (1 g) were added and the reaction stirred vigorouslyfor 30 min. Brine (2 mL) was added, the aqueous layer was saturated withsodium chloride and extracted with ethyl acetate (3×10 mL). Organiclayers were combined, dried and concentrated in vacuo to obtain 0.11 g(87%) of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(1-methylcarbonylamino)-(2,3,4-trihydroxy)butyl]cyclopentancarboxylate, MS (ES+) 547.5 [100%, (M+1)].

To a solution of the above solid (0.11 g, 0.2 mmol) in tetrahydrofuran(1 mL) was added 1 N sodium hydroxide (1.1 mL, 1.1 mmol) and the mixturestirred at room temperature for 2 h. Ether (2 mL) and water (2 mL) wereadded and organic layers were separated. The aqueous layer was washedwith ether (2×5 mL). The aqueous layer was acidified to pH 4 using 1 NHCl, saturated with sodium chloride and extracted with ethyl acetate(3×5 mL). The organic layers were combined, dried and concentrated invacuo to furnish 0.013 g (12%) of acid as a white solid, MS (ES+) 533.5[100%, (M+1)].

To a solution of the above acid (0.013 g, 0.24 mmol) in dichloromethane(2.0 mL) was added trifluoroacetic acid (0.04 mL, 0.48 mmol) and thereaction was stirred at room temperature overnight. Additionaltrifluoroacetic acid (0.02 mL, 0.24 mmol) was added and the reaction wasstirred at room temperature for 1 h. The solvent was removed in vacuoand traces of excess trifluoroacetic acid were removed in vacuo byco-distilling the residue twice with dichloromethane (10 mL). Theresidue was dissolved in water (1.0 mL), concentrated in vacuo and driedat acetone reflux temperature in vacuo to obtain 0.01 g of the titlecompound, mp 140-145° C.

Analysis: Calculated for C₁₃H₂₄N₄O₆.2.6C₂HF₃O₂: C, 34.76; H, 4.26; N,8.91 Found: C, 34.52; H, 4.20; N, 9.16

Example 16

c-3-{[(Amino)(imino)methyl]amino}-t-1-hydroxy-t-4-[(1-methylcarbonylamino)(2-trifluoromethyl-carbonyloxy)ethyl]cyclopentan-r-carboxylicacid trifluoroacetic acid (4:1) (isomer A at C-6)

To a mixture of tris-(methylthio)methane (1.08 g, 7.0 mmol) intetrahydrofuran (15 mL) at −78° C. was added n-butyllithium (1.6 M, 4.4mL, 7.0 mmol) under nitrogen over a period of 2 min. The mixture wasfurther stirred for 0.5 h at this temperature and to this was added amixture of3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxy-carbonyl)(methylcarbonylamino)methyl]cyclopentanone(Isomer A from Example 6, 0.49 g, 1.0 mmol) in tetrahydrofuran (5 mL).The reaction mixture was quenched with saturated ammonium chloridesolution (5 mL) after stirring for 1 h at −78° C. The mixture wasallowed to warm to room temperature and the organic layer was separated.The aqueous layer was further extracted with ether (10 mL). The combinedorganic layers were dried (MgSO₄), filtered and the filtrateconcentrated to give a syrup, which was purified by passing through acolumn of silica gel (25 g) using ether/hexane (3:1) as an eluent togive 0.42 g (66%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(ethoxycarbonyl)(methyl-carbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer A at C-6) as a white solid. An analytical sample was prepared byrecrystallization from ether/hexane, mp 155° C. (dec).

Analysis: Calculated for C₂₆H₄₆N₄O₈S₃: C, 48.88; H, 7.26; N, 8.77 Found:C, 49.00; H, 7.34; N, 8.64

To a solution of the above compound (1.5 g, 2.4 mmol) in tetrahydrofuran(25 mL) was added dropwise under nitrogen lithium borohydride (Aldrich,0.22 g, 9.6 mmol) and lithium 9-borabicyclo[3.3.1]nonane hydride(Aldrich, 1 M solution in tetrahydrofuran, 0.24 mL, 0.24 mmol) and thereaction mixture was stirred at room temperature overnight. More lithiumborohydride (0.16 g, 7.2 mmol) and lithium 9-borabicyclo[3.3.1]nonanehydride (Aldrich, 1 M solution in tetrahydrofuran, 0.24 mL, 0.24 mmol)was added and the reaction was stirred at room temperature for 4 h. Thereaction was quenched with 1 N sodium hydroxide (3 mL), brine (3 mL) andstirred for 5 min. The reaction was acidified to pH 4 using glacialacetic acid. Ether (10 mL) was added and the aqueous layer wasseparated. The aqueous layer was neutralized with saturated aqueoussodium bicarbonate and extracted with ether (2×10 mL). The organiclayers were combined, dried and concentrated in vacuo to obtain 1.55 gof a yellow oil. The oil was purified by flash column chromatography[20% chloroform:methanol:conc ammonium hydroxide (80:18:2) indichloromethane]. The oil was crystallized from ether/hexane to furnish0.6 g (42%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(2-hydroxy)(1-methyl-carbonylamino)ethyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer A at C-6) as a white solid, mp 108-112° C.

Analysis: Calculated for C₂₄H₄₄N₄O₇S₃: C, 48.30; H, 7.43; N, 9.39 Found:C, 48.37; H, 7.49; N, 9.25

To a mixture of the above compound (1.0 g, 1.69 mmol), mercuric chloride(1.69 g, 6.23 mmol) and mercuric oxide (0.48 g, 2.53 mmol) was addedmethanol/water (40/3 mL) and the reaction mixture was stirred at roomtemperature for 30 min. The mixture was filtered through a pad of Celiteand Florisil (17 g). The cake was washed with methanol (20 mL) and thefiltrate concentrated in vacuo to furnish 1.8 g of a white semisolid.The crude was purified by flash column chromatography [silica gel (33g); 75% ethyl acetate in hexane and 10% methanol in ethyl acetate] tofurnish 0.57 g (68%) of methylc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-cyclopentan-r-carboxylate(isomer A at C-6) as a white solid, mp 72-74° C. [R_(f)=0.47, 5%methanol in ethyl acetate].

Analysis: Calculated for C₂₂H₃₈N₄O₉: C, 52.58; H, 7.62; N, 11.15 Found:C, 52.85; H, 7.82; N, 10.93

To a solution of the above ester (0.5 g, 1.0 mmol) in tetrahydrofuran(10 mL) was added 1 N sodium hydroxide (2.0 mL, 2.0 mmol) and water (3mL). The reaction mixture was stirred at room temperature for 1 h,tetrahydrofuran was removed in vacuo and the aqueous layer was madeacidic with glacial acetic acid (pH=5). The aqueous layer was saturatedwith sodium chloride and extracted with ethyl acetate (5×10 mL). Theorganic layers were combined and concentrated in vacuo to furnish 0.43 g(88%) ofc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-cyclopentan-r-carboxylicacid (isomer A at C-6, as a white solid, MS (ES+) 489.5 [50%, (M+1)].

The above acid (0.29 g, 0.6 mmol) was dissolved in dichloromethane (12mL), trifluoroacetic acid (0.91 mL, 11.8 mmol) was added and thereaction was stirred at room temperature overnight. Additionaltrifluoroacetic acid (0.45 mL, 5.9 mmol) was added and the reaction wasstirred at room temperature for 1 h. The solvent was removed in vacuoand traces of excess trifluoroacetic acid were removed in vacuo byco-distilling the residue twice with dichloromethane (10 mL). Theresidue was triturated with ether to obtain 0.14 g (560%) of the titlecompound as a white solid, mp 148-160° C.

Analysis: Calculated for C₁₃H₁₉F₃N₄O₉.0.25C₂HF₃O₂: C, 39.28; H, 4.70; N,13.57 Found: C, 39.34; H, 5.00; N, 13.26

Example 17

c-3-{[(Amino)(imino)methyl]amino}-t-1-hydroxy-t-4-[(1-methylcarbonylamino)(2-trifluoromethyl-carbonyloxy)ethyl]cyclopentan-r-carboxylicacid trifluoroacetic acid (4:3) (isomer B at C-6)

To tris(methylthio)methane (Aldrich, 18.9 mL, 142 mmol) intetrahydrofuran (250 mL) at −78° C. was added dropwise over a period of10 min under nitrogen n-butyl lithium (1.6M solution in hexane, 98 mL,156 mmol) and stirred at −78° C. for 40 min. To the anion at −78° C. wasadded a solution of3β-{[(tert-butoxycarbonylamino)(t-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]cyclopentanone,isomer B from Example 6 (8.58 g, 17.7 mmol), in tetrahydrofuran (85 mL)dropwise over a period of 10 min and the reaction mixture stirred at−78° C. for 1.5 h. The reaction was quenched with saturated ammoniumchloride (50 mL) and warmed to room temperature. Ether (50 mL) was addedand the organic layers were combined, dried over MgSO₄ and concentratedin vacuo to furnish crude. Purification of the crude by flash columnchromatography (silica gel, 660 g, 30-50% ethyl acetate in hexane) gave3.8 g (34%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(ethoxycarbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer B at C-6) as a white solid, mp 94-96° C.

Analysis: Calculated for C₂₆H₄₈N₄O₈S₃: C, 48.88; H, 7.26; N, 8.77 Found:C, 49.08; H, 7.05; N, 8.75

To a solution of the above compound (1.4 g, 2.2 mmol) in tetrahydrofuran(22 mL) was added dropwise under nitrogen lithium borohydride (Aldrich,0.38 g, 16.43 mmol) and lithium 9-borabicyclo[3.3.1]nonane hydride(Aldrich, 1 M solution in tetrahydrofuran, 0.44 mL, 0.44 mmol), and thereaction mixture was stirred at room temperature overnight. The reactionwas quenched with 1 N sodium hydroxide (3 mL), brine (3 mL) and stirredfor 5 min. The reaction was acidified to pH 4 using glacial acetic acid,ether (10 mL) was added and the aqueous layer was separated. The aqueouslayer was neutralized with saturated aqueous sodium bicarbonate andextracted with ether (2×10 mL). The organic layers were combined, driedand concentrated in vacuo to obtain 1.44 g of a yellow oil. The oil waspurified by flash column chromatography [20% chloroform:methanol:concammonium hydroxide (80:18:2) in dichloromethane]. The oil wascrystallized from ether/hexane to furnish 0.47 g (36%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer B at C-6) as a white solid, mp 108-110° C.

Analysis: Calculated for C₂₄H₄₄N₄O₇S₃: C, 48.30; H, 7.43; N, 9.39 Found:C, 48.58; H, 7.51; N, 9.20

To a mixture of the above compound (0.78 g, 1.32 mmol), mercuricchloride (1.34 g, 4.9 mmol) and mercuric oxide (0.43 g, 1.97 mmol) wasadded methanol/water (29.5/2.5 mL) and the reaction mixture was stirredat room temperature for 30 min. The mixture was filtered through a padof Celite and Florisil (13 g). The cake was washed with methanol (20 mL)and the filtrate concentrated in vacuo to furnish 1.1 g of a whitesemisolid. The crude was purified by flash column chromatography [silicagel (30 g); 75% ethyl acetate in hexane and 10% methanol in ethylacetate] to furnish 0.42 g (630%) of methylc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-cyclopentan-r-carboxylate(isomer B at C-6) as a white solid, mp 194-198° C.

Analysis: Calculated for C₂₂H₃₈N₄O₉.0.5H₂O: C, 51.65; H, 7.68; N, 10.95Found: C, 51.37; H, 7.50; N, 10.93

To a solution of the above ester (0.38 g, 0.76 mmol) in tetrahydrofuran(7.5 mL) was added 1 N sodium hydroxide (1.5 mL, 1.5 mmol) and water(2.25 mL). The reaction mixture was stirred at room temperature for 1 h.Tetrahydrofuran was removed in vacuo and the aqueous layer was madeacidic with glacial acetic acid (pH=5). The aqueous layer was saturatedwith sodium chloride and extracted with ethyl acetate (5×10 mL). Theorganic layers were combined and concentrated in vacuo to furnish 0.325g (87%) of acid. This was triturated with ethyl acetate in hexane tofurnish 0.18 g ofc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-t-1-hydroxy-t-4-[(2-hydroxy)(1-methylcarbonylamino)ethyl]-cyclopentan-r-carboxylicacid (isomer B at C-6) as a white solid, MS (ES+) 489.4 [100%, (M+1)].

The above acid (0.15 g, 0.31 mmol) was dissolved in dichloromethane (6mL), trifluoroacetic acid (0.48 mL, 6.3 mmol) was added and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.24 mL, 3.2 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces ofexcess trifluoroacetic acid were removed in vacuo by co-distilling theresidue twice with dichloromethane (10 mL). The residue was trituratedwith ethanol/ether to obtain 0.125 g (86%) of the title compound as awhite solid, mp 210-220° C.

Analysis: Calculated for C₁₃H₁₉F₃N₄O₉.0.75C₂HF₃O₂: C, 37.07; H, 4.24; N,11.93 Found: C, 37.33; H, 4.45; N, 12.22

Example 18

t-3-Amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-hydroxycyclopentan-r-carboxylicacid trifluoroacetic acid ammonium trifluoroacetate (2:2:3)

A mixture ofc-3-(tert-butoxycarbonylamino)-t-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-[bis(phenylthio)methyl]cyclopentan-r-1-olfrom Example 8 (2.53 g, 4.1 mmol), mercuric oxide (1.90 g, 8.8 mmol),and boron trifluoride etherate (1.1 mL, 8.9 mmol) in a 15% aqueoussolution of tetrahydrofuran (70 mL) was stirred at room temperature for2 h. The reaction mixture was filtered through a pad of Celite andFlorisil. The filtrate was concentrated in vacuo to give 2.74 g of thecrude. To the above crude in methanol (50 mL) was added iodine (1.9 g,7.5 mmol) and the reaction mixture was heated to 50° C. To this mixturewas added dropwise a solution of potassium hydroxide (0.71 M/methanol,50 mL, 35.7 mmol). After 2 h stirring at 50° C., the reaction mixturewas filtered through Celite. The filtrate was concentrated in vacuo andthe resulting residue was dissolved in ethyl acetate (30 mL) and water(20 mL). The layers were separated and the aqueous layer was extractedwith ethyl acetate (4×10 mL). The combined organic extracts were washedwith brine, dried (MgSO₄), filtered through Celite, and concentrated invacuo to provide 3.6 g of crude. Purification by flash columnchromatography (silica gel, 200 g, 50-100% ethyl acetate/hexanes) gave0.224 g (14%) of the desired hydroxyacid as a white solid. To a solutionof hydroxyacid (0.244 g, 0.56 mmol) in dichloromethane (15 mL) was addedtrifluoroacetic acid (0.86 mL, 11.2 mmol) and the reaction mixture wasstirred at room temperature for 12 h. The reaction mixture wasconcentrated in vacuo to give crude. Purification by flash columnchromatography (silica gel, 60 g, chloroform/methanol/ammoniumhydroxide: 80/18/2) gave 0.241 g of a thick, yellow oil. Trituration ofthe yellow oil with ether provided 0.185 g (51%) of the title compoundas a tan solid, mp 37-39° C.

Analysis Calculated for C₁₇H₂₄N₂O₅.C₂HF₃O.1.5C₂H₄F₃NO₂: C, 40.84; H,4.83; N, 7.58 Found: C, 40.86; H, 5.08; N, 7.90

Example 19

c-3-{[(Amino)(imino)methyl]amino}-t-1-hydroxy-t-4{(methylcarbonylamino){[(methyl)-(methoxy)amino]carbonyl}methyl}cyclopentan-r-carboxylicacid trifluoroacetic acid (1:1)

To a solution oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-c-4-[(ethoxycarbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclo-pentan-r-ol(isomer B at C-6) (from Example 17, 2.1 g, 3.3 mmol) in ethanol (4 mL)and tetrahydrofuran (16.5 mL) was added 1 N sodium hydroxide (6.6 mL,6.6 mmol) and water (4 mL) and the reaction mixture stirred at roomtemperature for 2 h. Tetrahydrofuran was removed in vacuo and theaqueous layer was acidified to pH 5-4 using glacial acetic acid. Thesolid obtained was collected by filtration and dried in vacuo at toluenereflux temperature to furnish 1.78 g (87%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino-c-4-[(carboxy)-(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-olas a white solid, MS (ES+) 611.5.

To a solution of the above acid (0.92 g, 1.5 mmol) indichloromethane/tetrahydrofuran (12/3 mL) cooled to 0° C. was addedmethyl chloroformate (0.13 mL, 1.58 mmol) and triethylamine (0.25 mL,1.8 mmol). The reaction mixture was stirred at 0° C. for 30 min and acold prepared solution of N,O-dimethylhydroxylamine hydrochloride (0.22g, 2.25 mmol) and triethylamine (0.42 mL, 3.0 mmol) in dichloromethane(5 mL) that had been stirred at 0° C. for 30 min was added. The reactionmixture was allowed to warm to room temperature and stirred overnight.Ether (20 mL), tetrahydrofuran (5 mL) and 0.5 N sodium hydroxide (20 mL)were added and the organic layer was separated. The organic layer waswashed with brine (20 mL), dried and concentrated in vacuo to furnishcrude amide as an oil. Purification of the crude by flash columnchromatography (60 g silica gel, 50-100% ethyl acetate in hexane) gave0.63 g (64%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-{(methyl-carbonylamino){[(methoxy)(methyl)amino]carbonyl}methyl}-t-1-[tris(methylthio)methyl]-cyclopentan-r-olas a white solid. An analytical sample, mp 200° C., was prepared bycrystallization from ether.

Analysis: Calculated for C₂₆H₄₇N₅O₈S₃: C, 47.76; H, 7.25; N, 10.71Found: C, 47.96; H, 7.28; N, 10.63

To a mixture of the above compound (0.5 g, 0.77 mmol), mercuric chloride(0.78 g, 2.8 mmol) and mercuric oxide (0.25 g, 1.15 mmol) was addedmethanol/water (16.3/1.4 mL), and the reaction mixture stirred at roomtemperature for 30 min. The mixture was filtered through a pad of Celiteand Florisil (7 g). The cake was washed with methanol (20 mL) and thefiltrate concentrated in vacuo to furnish 1.8 g of a white semisolid.The crude was purified by flash column chromatography [silica gel (20g); 75% ethyl acetate in hexane and 10% methanol in ethyl acetate tofurnish 0.35 g (82%) of methylc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-t-4-{(methylcarbonylamino)-{[(methoxy)(methyl)amino]carbonyl}methyl}cyclopentan-r-carboxylateas a white solid, mp 72-74° C. [R_(f)=0.29 and 0.15, ethyl acetate]; MS(ES+) 560.6.

To a solution of above solid (0.352 g, 0.63 mmol) in tetrahydrofuran(6.3 mL) was added 1 N sodium hydroxide (1.3 mL, 1.3 mmol) and water (5mL) and the reaction mixture was stirred at room temperature for 1 h.Tetrahydrofuran was removed in vacuo and the aqueous layer was washedwith ether (2×10 mL). The aqueous layer was acidified to pH 5-4 usingglacial acetic acid, and saturated with sodium chloride and extractedwith ethyl acetate (3×10 mL). The combined organic layer was dried andconcentrated in vacuo to furnish 0.23 g (67%) ofc-4-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-1-hydroxy-t-4-{(methyl-carbonylamino){[(methoxy)(methyl)amino]carbonyl}methyl}cyclpentan-r-carboxylicacid as a white solid, MS (ES+) 546.6.

To a solution of above acid (0.18 g, 0.33 mmol) in dichloromethane (6.6mL) was added trifluoroacetic acid (0.51 mL, 6.6 mmol) and the reactionwas stirred at room temperature overnight. Additional trifluoroaceticacid (0.25 mL, 3.3 mmol) was added and the reaction was stirred at roomtemperature for 1 h. The solvent was removed in vacuo and traces ofexcess trifluoroacetic acid were removed in vacuo by co-distilling theresidue twice with dichloromethane (10 mL). The residue was dissolved inwater (5 mL) and concentrated in vacuo to obtain a white solid which wastriturated with ether to obtain 0.14 g (92%) of the title compound as awhite solid, mp 160-16° C.

Analysis: Calculated for C₁₃H₂₃N₅O₆C₂HF₂O₂: C, 39.22; H, 5.26; N, 15.25Found: C, 39.09; H, 5.29; N, 14.95

Example 20

3β-{[(Amino)(imino)methyl]amino}-4α-{{4-(methoxy)(methyl)amino]-1-(methylcarbonylamino)-2-oxo}butyl}cyclopentancarboxylicAcid Trifluoroacetic Acid (1:1)

To a solution of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethoxycarbonyl)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithianefrom Example 6 (6.74 g, 11.5 mmol) in ethanol (57.5 mL) andtetrahydrofuran (115 mL) was added 1 N sodium hydroxide (23 mL, 23 mmol)and water (35 mL) and the reaction mixture was stirred at roomtemperature for 5 h. Tetrahydrofuran was removed in vacuo and theaqueous layer was extracted with ethyl acetate (2×10 mL). The aqueouslayer was acidified to pH 5-4 using glacial acetic acid. The solidobtained was collected by filtration and dried in vacuo at acetonereflux temperature to furnish 5.95 g (93%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(carboxy)(methyl-carbonylamino)methyl]-1-cyclopentylidene}-1,3-dithianeas a white solid. An analytical sample, mp 158° C., was prepared bycrystallization from ethanol.

Analysis: Calculated for C₂₄H₃₈N₄O₇S₂.0.75C₂H₆O: C, 51.63; H, 7.22; N,9.44 Found: C, 51.70; H, 7.26; N, 9.18

To a solution of the above acid (0.63 g, 1.13 mmol) indichloromethane/tetrahydrofuran (8/2 mL) cooled to 0° C. was addedmethyl chloroformate (0.1 mL, 1.24 mmol) and triethylamine (0.19 mL,1.36 mmol). The reaction mixture was stirred at 0° C. for 30 min and acold solution of N,O-dimethylhydroxylamine hydrochloride (0.17 g, 1.7mmol) and triethylamine (0.32 mL, 2.26 mmol) in dichloromethane (5 mL)that had been stirred at 0° C. for 30 min was added. The reactionmixture was allowed to warm to room temperature and stirred overnight.Brine (5 mL), water (5 mL) and saturated sodium carbonate (5 mL) wereadded and the organic layer was separated. The organic layer was driedand concentrated in vacuo to furnish an oil. Purification of the crudeby flash column chromatography (34 g silica gel, 50-75% ethyl acetate inhexane) gave 0.48 g (63%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-{{[(methoxy)(methyl)amino]carbonyl}methyl}cyclopentylidene}-1,3-dithiane.An analytical sample, mp 190-192° C., was prepared by crystallizationfrom ether/hexane.

Analysis: Calculated for C₂₆H₄₃N₅O₇S₂: C, 51.89; H, 7.20; N, 11.64Found: C, 52.32; H, 7.24; N, 11.33

To a solution of the above compound (3.4 g, 5.7 mmol) in tetrahydrofuran(50 mL) was added dropwise vinylmagnesium bromide (Aldrich, 1 M solutionin tetrahydrofuran, 33.94 mL, 33.94 mmol) over a period of 10 min. Thereaction mixture was stirred at room temperature for 10 min and quenchedwith saturated ammonium chloride solution and brine (1:1, 30 mL). Ether(25 mL) was added and the organic layer was separated. The aqueous layerwas washed with ether (25 mL). The organic layers were combined, driedand concentrated in vacuo to furnish an oil. Purification of the crudeby flash column chromatography (180 g silica gel, 20-70% ethyl acetatein hexane) gave 2.0 g (56%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-{{4-[(methoxy)(methyl)amino]-1-(methylcarbonyl-amino)-2-oxo}butyl}cyclopentylidene}-1,3-dithianeas a white solid, mp 99-101° C.

Analysis: Calculated for C₂₈H₄₇N₅O₇S₃: C, 53.40; H, 7.52; N, 11.12; S,10.18 Found: C, 53.81; H, 7.48; N, 10.96; S, 10.25

To a solution of the above compound (0.55 g, 0.9 mmol) in methanol (26.4mL) was added 6 N HCl (2.2 mL, 13.0 mmol) and the mixture was stirred atroom temperature until all starting material had disappeared (TLCanalysis, ethyl acetate, ˜30 h). The reaction mixture was cooled to 0°C. and sodium hydroxide (0.72 g, 18 mmol) was added and the reaction wasstirred at room temperature for 1 h. The reaction was quenched withglacial acetic acid (0.5 mL) and concentrated in vacuo to furnish aresidue. To the residue was added ethyl acetate (10 mL) and water (10mL). The aqueous layer was separated and extracted with ethyl acetate(10 mL). The organic layers were combined and concentrated in vacuo tofurnish crude. The above crude was purified by flash columnchromatography (50-80% ethyl acetate in hexane) to furnish 0.29 g (56%)of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-{{4-[(methoxy)-(methyl)amino]-1-(methylcarbonylamino)-2-oxo}butyl}cyclopentancarboxylateas an oil. The oil was crystallized from ether as a white solid.

Analysis: Calculated for C₂₆H₄₅N₅O₉: C, 54.63; H, 7.93; N, 12.25 Found:C, 54.66; H, 7.87; N, 11.94

To a solution of the above ester (0.2 g, 0.35 mmol) in tetrahydrofuran(3.5 mL) was added 1 N sodium hydroxide (0.88 mL, 0.88 mmol) and water(2 mL) and the reaction mixture was stirred at room temperature for 2 h.Tetrahydrofuran was removed in vacuo and water (5 mL) was added. Theaqueous layer was washed with ether (2×5 mL) and acidified to pH 5-4using glacial acetic acid. The aqueous layer was saturated with sodiumchloride and extracted with ethyl acetate (3×10 mL). The combinedorganic layers were dried and concentrated in vacuo to furnish 0.18 g ofthe corresponding cyclopentancarboxylic acid as a white solid, MS (ES+)558.3 [100%, (M+1)].

To a solution of the above acid (0.18 g, 0.33 mmol) in dichloromethane(6.6 mL) was added trifluoroacetic acid (0.51 mL, 6.6 mmol) and thereaction was stirred at room temperature overnight. Additionaltrifluoroacetic acid (0.25 mL, 3.3 mmol) was added and the reaction wasstirred at room temperature for 1 h. The solvent was removed in vacuoand traces of excess trifluoroacetic acid were removed in vacuo byco-distilling the residue twice with dichloromethane (10 mL). Theresidue was washed with ether (2×10 mL), precipitated withmethanol/ether and dried at toluene reflux temperature in vacuo toobtain the title compound as a tan solid, mp 189-192° C.

Analysis: Calculated for C₁₅H₂₇N₅O₅.C₂HF₃O₂: C, 43.31; H, 5.99; N, 14.85Found: C, 43.38; H, 5.74; N, 14.42

Example 21

t-3-{[(Amino)(imino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid trifluoroacetic acid (3:5)

To a stirred solution of tris(methylthio)methane (1.6 mL, 12 mmol) intetrahydrofuran (20 mL) at −78° C. was added dropwise n-butyl lithium(2.5M, 5.3 mL, 13.3 mmol). After 30 min of stirring,3β-tert-butoxycarbonylamino-4α-[bis(ethoxycarbonyl)(methylcarbonylamino)methyl]-cyclopentanonefrom Example 8 (1.0 g, 2.4 mmol) in tetrahydrofuran (15 mL) was addeddropwise. The reaction mixture was stirred at −78° C. for 3 h thenquenched with a saturated aqueous solution of ammonium chloride (15 mL).The separated aqueous layer was extracted with ether (4×10 mL). Thecombined organic extracts were washed with brine, dried (MgSO₄),filtered through Celite, and concentrated in vacuo to provide crude.Purification by radial PLC (silica gel, 25-35% ethyl acetate/hexane)gave 0.48 g (35%) ofc-3-(tert-butoxycarbonylamino)-t-4-[bis-(ethoxycarbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-olas a white solid, mp 98-100° C.

Analysis: Calculated for C₂₃H₄₀N₂O₈S₃: C, 48.57; H, 7.09; N, 4.93 Found:C, 48.74; H, 7.00; N, 4.91

To a mixture of the above compound (1.71 g, 3.0 mmol) in ethanol (15 mL)was added 1 N sodium hydroxide (15 mL) and heated at reflux for 2 h. Themixture was acidified with acetic acid and heated at reflux again for 1h and concentrated. To the residue was added water (50 mL) and themixture extracted with dichloromethane (3×50 mL). The combined organicextracts were dried (MgSO₄), filtered and concentrated to givec-3-(tert-butoxycarbonylamino)-t-4-[(carboxy)(methylcarbonylamino)methyl]-t-1-[tris(methylethio)methyl]cyclopentan-r-ol(1.2 g, 85%).

To a mixture of the above acid (1.2 g, 2.56 mmol) in tetrahydrofuran (15mL) at −5° C. was added triethylamine (0.29 g 2.8 mmol) and ethylchloroformate (0.31 g, 2.8 mmol) and stirred for 0.5 h. To this mixturewas then added diethylamine (0.38 g, 5.2 mmol) and stirred at 0° C. for1 h and at room temperature for 3 h. The mixture was diluted with ethylacetate (100 mL) and water (75 mL). The organic layer was separated,washed with water (100 mL) and brine (100 mL), and dried (MgSO₄). Afterfiltration, the filtrate was concentrated to give 1.1 g (82%) of crudec-3-(tert-butoxycarbonylamino)-t-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol.

To a mixture of the above crude amide (1.10 g, 2.10 mmol) in a methanol(12):water (1) mixture (51.0 mL) was added mercury (II) chloride (2.10g, 7.75 mmol) and mercury (II) oxide (0.69 g, 3.18 mmol) and stirred for2 h. The solids were removed by filtration through Celite and washedwith dichloromethane (100 mL). To the filtrate was added water (100 mL)and the organic layer separated. The aqueous layer was further extractedwith dichloromethane (2×80 mL). The combined organic layers were dried(MgSO₄), filtered and the filtrate concentrated to give 0.9 g (100%) ofa syrup of crude methylt-3-(tert-butoxycarbonylamino)-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylate.

A mixture of the above crude ester (0.9 g) in dichloromethane (50 mL)was stirred with trifluoroacetic acid (5.0 mL) for 16 h. The reactionmixture was concentrated and dried in vacuo to give 0.93 g (100%) of thecorresponding crude methylt-3-amino-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylate.It was used as such for the next step.

To a mixture of above amine (0.93 g, 2.1 mmol) in dimethylformamide (20mL) were added triethylamine (1.06 g, 10.5 mmol),N,N′-bis-tert-butoxycarbonyl-S-methylisothiourea (0.61 g, 2.1 mmol), andmercury (II) chloride (0.57 g, 2.1 mmol) and the mixture stirred at roomtemperature for 2 h. The mixture was diluted with ethyl acetate (100 mL)and filtered through Celite. The filtrate was washed with water (2×100mL), and brine (1×100 mL). The organic layer was dried (MgSO₄), filteredand concentrated to give a syrup, which was purified by passing througha column of silica gel (50 g) using 5% methanol in ethyl acetate to give0.65 g (54%) of methylt-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylateas a white powder, mp >120° C. (dec).

Analysis: Calculated for C₂₆H₄₅N₅O₉: C, 54.63; H, 7.93; N, 12.25 Found:C, 54.56; H, 7.97; N, 12.04

A mixture of the above ester (0.88 g, 0.66 mmol) in 0.1 N sodiumhydroxide (13.0 mL, water-1, tetrahydrofuran-1, ethanol-1 mixture) wasstirred at room temperature for 2 h. It was neutralized with acetic acidafter filtration through a cotton plug and stirred at room temperaturefor 16 h. The precipitate obtained was collected by filtration, washedwith water and dried in vacuo to give 0.33 g (90%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid as a white solid, mp >235° C. (dec).

Analysis: Calculated for C₂₅H₄₃N₅O₉: C, 53.85; H, 7.77; N, 12.56 Found:C, 53.74; H, 7.83; N, 12.56

A mixture oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid (0.9 g, 0.16 mmol) in dichloromethane (5.0 mL) was stirred withtrifluoroacetic acid (0.5 mL) for 48 h. It was then concentrated anddried in vacuo to give 0.8 g (90%) the title compound as a brown powder,mp 99-103° C. (dec).

Analysis: Calculated for C₁₅H₂₇N₅O₅.1.67C₂HF₃O₂: C, 40.23; H, 5.27; N,12.79 Found: C, 40.38; H, 5.28; N, 12.38

Example 22

t-3-Amino-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxy-cyclopentan-r-carboxylicacid trifluoroacetic acid (1:1) (isomer A at C-6)

To a mixture ofc-3-(tert-butoxycarbonylamino)-t-4-[bis(ethoxycarbonyl)-(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-olfrom Example 21 (1.46 g, 2.6 mmol) in ethanol (20 mL) and water (10 mL)was added 1 N sodium hydroxide (10 mL, 10 mmol) and heated at reflux for2 h. The mixture was concentrated in vacuo and acidified with glacialacetic acid (1.0 mL, 17.5 mmol). To the concentrate was added ethylacetate (20 mL) and heated at reflux for 1 h. The layers were separatedand the aqueous layer was extracted with ether (4×10 mL). The combinedorganic extracts were washed with brine, dried (MgSO₄), filtered throughCelite, and concentrated in vacuo to providec-3-(tert-butoxycarbonylamino)-t-4-[(carboxy)(methylcarbonylamino)methyl]-t-1-[tris(methyl-thio)methyl]cyclopentan-r-ol(0.98 g, 76%).

To a stirred mixture of the above acid (0.97 g, 2.0 mmol) intetrahydrofuran (25 mL) at 0° C. was added ethyl chloroformate (2.1 mL,2.2 mmol) and triethylamine (0.35 mL, 2.5 mmol). After stirring for 20min, the reaction mixture was allowed to warm to room temperature,stirred for an additional 30 min, and filtered through Celite. Thefiltrate was concentrated in vacuo to give 0.85 g (100%) of crude mixedanhydride.

To a mixture of the above mixed anhydride (0.84 g, 1.56 mmol) intetrahydrofuran (20 mL) at 0° C. was added di-n-propylamine (0.6 mL, 4.4mmol). The reaction mixture was stirred at 0° C. for 30 min and at roomtemperature for 3 h. The mixture was diluted with water (10 mL) andlayers were separated. The aqueous layer was extracted with ethylacetate (4×10 mL). The combined organic extracts were washed with brine,dried (MgSO₄), filtered through Celite, and concentrated in vacuo toafford 1.0 g of the crude. Purification by radial PLC (silica gel,35-50% ethyl acetate/hexane) gave 0.23 g (27%) ofc-3-(tert-butoxycarbonylamino)-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer A at C-6) as a single isomer.

To a mixture of the above amide (0.17 g, 0.31 mmol) in methanol (16.5mL) and water (1.5 mL) at room temperature was added mercuric oxide(0.10 g, 0.47 mmol) and mercuric chloride (0.31 g, 1.2 mmol). Afterstirring for 2 h, the reaction mixture was filtered through a pad ofFlorisil and Celite. The filtrate was concentrated in vacuo to give 0.26g of crude. Purification by radial PLC (silica gel, 50-75% ethylacetate/hexane) furnished 0.14 g (96%) of methylt-3-(tert-butoxycarbonylamino)-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylate.

To a mixture of the above ester (0.13 g, 0.29 mmol) in tetrahydrofuran(3.5 mL) and water (2.5 mL) at room temperature was added 1 N sodiumhydroxide (0.6 mL, 0.6 mmol). The reaction mixture was stirred for 1 hand concentrated in vacuo. The concentrate was acidified to pH 5-4 withglacial acetic acid and extracted with ethyl acetate (5×10 mL). Thecombined organic extracts were washed with brine, dried (MgSO₄),filtered through Celite, and concentrated in vacuo to afford 0.13 g(100%) of crudet-3-(tert-butoxycarbonylamino)-c-3-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid.

A mixture of the above crude acid (0.13 g, 0.29 mmol) in dichloromethane(10 mL) with trifluoroacetic acid (0.45 mL, 5.8 mmol) was stirred atroom temperature overnight. The reaction mixture was concentrated invacuo to give 0.16 g of a thick oil which was triturated with ether toprovide 0.083 g (63%) of the title compound as a tan solid, mp 168-170°C.

Analysis: Calculated for C₁₆H₂₉N₃O₅.C₂HF₃O₂: C, 47.26; H, 6.61; N, 9.19Found: C, 47.47; H, 6.83; N, 9.33

Example 23

t-3-{[(Amino)(imino)methyl]amino}-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-hydroxycyclpentan-r-carboxylicacid trifluoroacetic acid (4:5)

To a mixture ofc-3-(tert-butoxycarbonylamino)-t-4-[bis(ethoxycarbonyl)(methyl-carbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-olfrom Example 21 (1.46 g, 2.6 mmol) in ethanol (20 mL) and water (10 mL)was added 1 N sodium hydroxide (10 mL, 10 mmol) and heated at reflux for2 h. The mixture was concentrated in vacuo and acidified with glacialacetic acid (1.0 mL, 17.5 mmol). To the concentrate was added ethylacetate (20 mL) and heated at reflux for 1 h. The layers were separatedand the aqueous layer was extracted with ether (4×10 mL). The combinedorganic extracts were washed with brine, dried (MgSO₄), filtered throughCelite, and concentrated in vacuo to providec-3-(tert-butoxycarbonylamino)-t-4-[(carboxy)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(0.98 g, 76%).

To a stirred mixture of above solid (0.97 g, 2.0 mmol) intetrahydrofuran (25 mL) at 0° C. was added ethyl chloroformate (0.21 mL,2.2 mmol) and triethylamine (0.35 mL, 2.5 mmol). After stirring for 20min, the reaction mixture was allowed to warm to room temperature,stirred for an additional 30 min, and filtered through Celite. Thefiltrate was concentrated in vacuo to give 0.85 g (100%) of the crudemixed anhydride.

To a mixture of above mixed anhydride (0.84 g, 1.56 mmol) intetrahydrofuran (20 mL) at 0° C. was added di-n-propylamine (0.6 mL, 4.4mmol). The reaction mixture was stirred at 0° C. for 30 min and at roomtemperature for 3 h. The mixture was diluted with water (10 mL) and thelayers were separated. The aqueous layer was extracted with ethylacetate (4×10 mL) The combined organic extracts were washed with brine,dried (MgSO₄), filtered through Celite, and concentrated in vacuo toafford 1.0 g of crude. Purification by radial PLC (silica gel, 35-50%ethyl acetate/hexane) gave 0.58 g (68%) ofc-3-(tert-butoxycarbonylamino)-t-3-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-t-1-[tris(methylthio)methyl]cyclopentan-r-ol.

To a mixture of the above amide (0.34 g, 0.63 mmol) in methanol (16.5mL) and water (1.5 mL) at room temperature was added mercuric oxide(0.210 g, 0.97 mmol) and mercuric chloride (0.64 g, 2.4 mmol). Afterstirring for 2 h, the reaction mixture was filtered through a pad ofFlorisil and Celite. The filtrate was concentrated in vacuo to give 0.44g of crude. Purification by radial PLC (silica gel, 50% ethylacetate/hexane) furnished 0.27 g (94%) of methylt-3-(tert-butoxycarbonylamino)-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylate.

A mixture of the above ester (0.15 g, 0.32 mmol) in dichloromethane (10mL) with trifluoroacetic acid (0.5 mL, 6.6 mmol) was stirred at roomtemperature overnight. The reaction mixture was concentrated in vacuo togive 0.16 g of methylt-3-amino-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylateas a thick oil.

To a mixture of above amine (0.16 g, 0.33 mmol) in dimethylformamide (3mL) was added N,N′-bis-tert-butoxycarbonyl-S-methyl isothiourea (0.11 g,0.37 mmol), triethylamine (0.3 mL, 2.2 mmol), and mercuric chloride(0.10 g, 0.37 mmol). The reaction mixture was stirred at roomtemperature for 3 h. To this mixture was added water (5 mL) and thelayers were separated. The organic layer was washed with brine, dried(MgSO₄), filtered through Celite, and concentrated in vacuo to afford0.22 g of a yellow solid. Purification by radial PLC (silica gel, 50-75%ethyl acetate/hexane) gave 0.13 g (67%) of methylt-3{[(tert-butoxycarbonylamino)(tert-butoyxcarbonylimino)methyl]amino}-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylate.

To a mixture of the above compound (0.13 g, 0.22 mmol) intetrahydrofuran (4 mL) and water (2 mL) at room temperature was added 1N sodium hydroxide (0.5 mL, 0.5 mmol). The reaction mixture was stirredfor 2 h and concentrated in vacuo. The concentrate was acidified to pH5-4 with glacial acetic acid and extracted with ethyl acetate (5×10 mL).The combined organic extracts were washed with brine, dried (MgSO₄),filtered through Celite, and concentrated in vacuo to afford 0.13 g(100%) of crudet-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylicacid.

A mixture of the above acid (0.13 g, 0.22 mmol) and trifluoroacetic acid(0.5 mL, 6.6 mmol) in dichloromethane (10 mL) was stirred at roomtemperature overnight. The reaction mixture was concentrated in vacuo togive 0.2 g of the crude. Trituration with ether afforded 0.08 g (74%) ofthe title compound as a tan solid, mp 153-155° C.

Analysis: Calculated for C₁₇H₃₁N₅O₅.1.25C₂HF₃O₂: C, 44.36; H, 6.16; N,13.26 Found: C, 44.34; H, 6.17; N, 13.19

Example 24

c-3-{[(Amino)(imino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylicacid trifluoroacetic acid (1:1) (isomer A at C-6)

To a mixture oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-c-4-[(ethoxycarbonyl)(methlycarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclo-pentan-r-ol(isomer A at C-6) from Example 16 (2.55 g, 4.0 mmol) in tetrahydrofuran(20 mL) and ethanol (10 mL) was added 1 N aqueous sodium hydroxide (7.0mL, 7.0 mmol) and stirred at room temperature for 4 h. Afterneutralization with acetic acid, the mixture was concentrated. To theresidue was added water (50 mL) and stirred for 4 h. The whiteprecipitate obtained was collected by filtration, washed with water anddried in vacuo at 60° C. for 24 h to give 2.1 g (86%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(carboxy)(methyl-carbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(mixture of isomers) as a white solid, mp 228-230° C. (dec).

Analysis: Calculated for C₂₄H₄₂N₄O₈S₃.H₂O: C, 45.84; H, 7.05; N, 8.91Found: C, 45.31; H, 6.64; N, 9.05

To a mixture of the above acid (0.61 g, 1 mmol) in tetrahydrofuran (5mL) at −5° C. was added triethylamine (0.1 g, 1 mmol) and ethylchloroformate (0.11 g, 1 mmol) and stirred for 0.5 h. To this mixturewas then added di-n-propylamine (0.1 g, 1 mmol), the mixture was stirredat 0° C. for 1 h and at room temperature for 1 h. The mixture wasdiluted with ethyl acetate (40 mL) and water (40 mL). The organic layerwas separated, washed with water (50 mL) and brine (50 mL), and dried(MgSO₄). After filtration the filtrate was concentrated and the residuepassed through a column of silica gel (25 g) using ethyl acetate/hexane(1:1) as an eluent to give 0.37 g (53%) oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-c-4-[(di-n-propylamino-carbonyl)(methylcarbonylamino)methyl]-t-1-[tris(methylthio)methyl]cyclopentan-r-ol(isomer A at C-6) as a white solid, mp 88-90° C.

Analysis: Calculated for C₃₀H₅₅N₅O₇S₃: C, 51.92; H, 7.99; N, 10.09Found: C, 52.15; H, 8.04; N, 9.95

To a mixture of the above amide (0.6 g, 0.9 mmol) in methanol (12):water(1) mixture (22.0 mL) was added mercury (II) chloride (0.91 g, 3.35mmol) and mercury (II) oxide (0.3 g, 1.40 mmol) and stirred for 0.5 h.The solids were removed by filtration through Celite and washed withdichloromethane (50 mL). To the filtrate was added water (50 mL) and anorganic layer separated. The aqueous layer was further extracted withdichloromethane (2×40 mL). The combined organic layers were dried(MgSO₄), filtered and the filtrate concentrated to give a syrup, whichwas purified by passing through a column of silica gel (50 g) usingethyl acetate as an eluent to give 0.07 g (13%) of methylc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylamino)-methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylate(isomer A at C-6) as a white solid, mp 128-130° C. (dec).

Analysis: Calculated for C₂₈H₄₉N₅O₉: C, 56.08; H, 8.23; N, 11.68 Found:C, 56.20; H, 8.10; N, 11.84

A mixture of the above ester (0.13 g, 0.22 mmol) in tetrahydrofuran (2.0mL) and ethanol (1.0 mL) was stirred with 1 N sodium hydroxide (0.5 mL,0.5 mmol) for 1 h. It was neutralized with acetic acid and theprecipitate obtained was collected by filtration, washed with water anddried in vacuo to give 0.08 g (67%) ofc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylicacid (isomer A at C-6) as an off-white solid, mp 225-230° C. (dec).

Analysis: Calculated for C₂₇H₄₇N₅O₉.0.5H₂O: C, 54.53; H, 8.13; N, 11.78Found: C, 54.25; H, 7.90; N, 11.48

A mixture of the above acid (0.66 g, 0.10 mmol) in dichloromethane (4.0mL) was stirred with trifluoroacetic acid (1.0 mL) for 16 h. It was thenconcentrated and dried in vacuo to give 0.04 g (800) of the titlecompound as a white solid, mp 128-130° C. (dec).

Analysis: Calculated for C₁₇H₃₁N₅O₅.C₂HF₃O₂: C, 45.69; H, 6.46; N, 14.02Found: C, 46.37; H, 6.69; N, 14.13

Example 25

c-3-{[(Amino)(imino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-hydroxycyclopentan-r-carboxylicacid trifluoroacetic acid (1:1) (isomer B at C-6)

To a mixture oft-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-t-1-[tris(methylthio)-methyl]cyclopentan-r-ol(isomer B at C-6) which was isolated as a by-product from thepreparation of isomer A in Example 24 (0.51 g, 0.73 mmol) in methanol(12):water (1) mixture (18.0 mL) was added mercury (II) chloride (0.75g, 2.75 mmol) and mercury (II) oxide (0.24 g, 1.12 mmol) and stirred for2 h. The solids were removed by filtration through Celite and washedwith dichloromethane (50 mL). To the filtrate was added water (50 mL)and an organic layer separated. The aqueous layer was further extractedwith dichloromethane (2×40 mL). The combined organic layers were dried(MgSO₄), filtered and the filtrate concentrated to give a white solid,which was recrystallized from ether/hexane to give 0.35 g (80%) ofmethylc-3-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)-(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylate(isomer B at C-6) as a white solid, mp 170-172° C. (dec).

Analysis: Calculated for C₂₈H₄₉N₅O₉: C, 56.08; H, 8.23; N, 11.68 Found:C, 55.96; H, 8.29; N, 11.70

To mixture of the above ester (0.25 g, 0.42 mmol) in tetrahydrofuran(3.0 mL) and ethanol (1.3 mL) was stirred with 1 N sodium hydroxide (1.0mL, 1.0 mmol) for 2 h. The solvent was evaporated and the residue wasdissolved in water (1 mL) and neutralized with acetic acid. Theprecipitate obtained was collected by filtration, washed with water anddried in vacuo to give 0.20 g (79%) of the corresponding acid.

A mixture of above acid (0.15 g, 0.26 mmol) in dichloromethane (10 mL)was stirred with trifluoroacetic acid (1.2 mL) for 16 h. It was thenconcentrated and dried in vacuo to give 0.11 g (85%) of the titlecompound as a white solid, mp 202-205° C. (dec).

Analysis: Calculated for C₁₇H₃₁N₅O₅.C₂HF₃O₂: C, 45.69; H, 6.46; N, 14.02Found: C, 45.84; H, 6.51; N, 13.82

Example 26

3β-{[(Amino)(imino)methyl]amino}-4α-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-cyclopentancarboxylicacid (isomer A at C-6)

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithianefrom Example 20 (0.56 g, 1 mmol) in tetrahydrofuran (8 mL) at 0° C. wasadded triethylamine (0.11 g, 1.1 mmol) and methyl chloroformate (0.1 g,1.1 mmol) and stirred for 0.5 h. To this mixture was then addeddiethylamine (0.11 g, 1.5 mmol) and stirred at 0° C. for 1 h and at roomtemperature for 1 h. The mixture was diluted with ethyl acetate (40 mL)and water (40 mL). The organic layer was separated, washed with water(50 mL) and brine (50 mL), and dried (MgSO₄). After filtration, thefiltrate was concentrated and the residue passed through a column ofsilica gel (50 g) using ethyl acetate/hexane (1:1) as an eluent to give0.21 g (34%) of the desired2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(diethylaminocarbonyl)-(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithianeas a white solid.

A mixture of the above amide (0.1 g, 0.016 mmol) in 0.5 N HCl inmethanol (5.0 mL, 2.5 mmol) was stirred for 24 h at room temperature and2 h at 45° C. To the mixture was added 6.0 N HCl (0.2 mL, 1.2 mmol) andheated at 45° C. for another 2 h. The reaction mixture was thenconcentrated and the residue stirred with 0.1 N sodium hydroxide (5.0mL, 0.5 mmol) for 1 h, concentrated, and again stirred with 1 N sodiumhydroxide (1.0 mL, 1.0 mmol) for 0.5 h. It was then filtered through acotton plug and neutralized with dilute hydrochloric acid to give thetitle compound mixed with sodium chloride, MS (ES+) 342.3 (M+1, 100%).

Example 27

3β-{[(Amino)(imino)methyl]amino}-4α-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]-cyclopentancarboxylicacid (isomer A at C-6)

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer A) from Example 20 (0.56 g, 1 mmol) in tetrahydrofuran (8 mL) at0° C. was added triethylamine (0.11 g, 1.1 mmol) and methylchloroformate (0.1 g, 1.1 mmol) and stirred for 0.5 h. To this mixturewas then added di-n-propylamine (0.15 g, 1.5 mmol) and stirred at 0° C.for 1 h and at room temperature for 1 h. The mixture was diluted withethyl acetate (40 mL) and water (40 mL). The organic layer wasseparated, washed with water (50 mL) and brine (50 mL), and dried(MgSO₄). After filtration, the filtrate was concentrated and the residuepassed through a column of silica gel (50 g) using ethyl acetate/hexane(1:1) as an eluent to give 0.22 g (34%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(di-n-propylaminocarbonyl)-(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer A) as a white solid, mp 125-126° C.

Analysis: Calculated for C₃₀H₅₁N₅O₆S₂: C, 56.14; H, 8.01; N, 10.91Found: C, 56.72; H, 8.05; N, 10.76

A mixture of the above compound (0.11 g, 0.016 mmol) in 0.5 N HCl inmethanol (5.0 mL, 2.5 mmol) was stirred for 24 h at room temperature and2 h at 45° C. To the mixture was further added 6.0 N hydrochloric acid(0.2 mL, 1.2 mmol) and heated at 45° C. for another 2 h. The reactionmixture was then concentrated and the residue stirred with 0.1 N sodiumhydroxide (5.0 mL, 0.5 mmol) for 1 h, concentrated, and again stirredwith 1 N sodium hydroxide (1.0 mL, 1.0 mmol) for 0.5 h. It was thenfiltered through a cotton plug, neutralized with dilute hydrochloricacid and concentrated to give the title compound mixed with sodiumchloride, MS (ES+) 370.4 (M+1, 100%).

Example 28

3β-{[(Amino)(imino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]-cyclopentancarboxylicacid (isomer A at C-6)

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentyl-idine}-1,3-dithiane(isomer A) from Example 20 (0.56 g, 1 mmol) in tetrahydrofuran (8 mL) at0° C. was added triethylamine (0.11 g, 1.1 mmol) and methylchloroformate (0.1 g, 1.1 mmol) and stirred for 0.5 h. To this mixturewas then added 3-pentylamine (0.2 g, 2.3 mmol) and stirred at 0° C. for1 h and at room temperature for 1 h. The mixture was diluted with ethylacetate (40 mL) and water (40 mL). The organic layer was separated,washed with water (50 mL) and brine (50 mL), and dried (MgSO₄). Afterfiltration, the filtrate was concentrated and the residue passed througha column of silica gel (50 g) using ethyl acetate/hexane (1:1) as aneluent to give 0.28 g (45%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer A at C-6) as a white solid, mp >230° C. (dec).

Analysis: Calculated for C₂₉H₄₉N₅O₆S₂: C, 55.48; H, 7.86; N, 11.15Found: C, 55.96; H, 7.92; N, 10.99

A mixture of the above compound (0,1 g, 0.016 mmol) in 0.5 N HCl inmethanol (5.0 mL, 2.5 mmol) was stirred for 24 h at room temperature and2 h at 45° C. To the mixture was further added 6.0 N hydrochloric acid(0.2 mL, 1.2 mmol) and heated at 45° C. for another 2 h. The reactionmixture was then concentrated and the residue stirred with 0.1 N sodiumhydroxide (5.0 mL, 0.5 mmol) for 1 h, concentrated, and again stirredwith 1 N sodium hydroxide (1.0 mL, 1.0 mmol) for 0.5 h. It was thenfiltered through a cotton plug, neutralized with dilute hydrochloricacid and concentrated to give the title compound mixed with sodiumchloride, MS (ES+) 356.5 (M+1, 100%).

Example 29

3β-{[(Amino)(imino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)-methyl]cyclopentancarboxylicacid (isomer B at C-6)

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithiane (isomer A) from Example 20(0.56 g, 1 mmol) in tetrahydrofuran (8 mL) at 0° C. was addedtriethylamine (0.11 g, 1.1 mmol) and methyl chloroformate (0.1 g, 1.1mmol) and stirred for 0.5 h. To this mixture was then added3-pentylamine (0.2 g, 2.3 mmol) and stirred at 0° C. for 1 h and at roomtemperature for 1 h. The mixture was diluted with ethyl acetate (40 mL)and water (40 mL). The organic layer was separated, washed with water(50 mL) and brine (50 mL), and dried (MgSO₄). After filtration, thefiltrate was concentrated and the residue passed through a column ofsilica gel (50 g) using ethyl acetate/hexane (1:1) as an eluent to give0.06 g (10%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer B at C-6) as a white solid, mp >200° C. (dec).

Analysis: Calculated for C₂₉H₄₉N₅O₆S₂: C, 55.48; H, 7.86; N, 11.15Found: C, 55.21; H, 7.72; N, 11.06

To a mixture of the above compound (isomer B, 0.035 g, 0.005 mmol) in0.5 N HCl in methanol (3.0 mL, 1.5 mmol) was stirred for 24 h at roomtemperature and 2 h at 45° C. To the mixture was further added 6.0 Nhydrochloric acid (0.2 mL, 1.2 mmol) and heated at 45° C. for another 2h. The reaction mixture was then concentrated and stirred with 1 Nsodium hydroxide (0.4 mL, 0.4 mmol) for 4 h. It was then filteredthrough a cotton plug, neutralized with dilute hydrochloric acid andconcentrated to give the title compound mixed with sodium chloride, MS(ES+) 356.4 (M+1, 100%).

Example 30

3β-{[(Amino)(imino)methyl]amino}-4α-[(diethylaminocarbonyl)(methylcarbonylamino)-methyl]cyclopentancarboxylicacid (isomer A at C-6, isomer A at C-1)

To a mixture of2-{3β-(tert-butoxycarbonylamino)-4α-[(carboxy)(methyl-carbonylamino)methyl]cyclopentylidene}-1,3-dithiane(from Example 10) (10 g, 24.0 mmol) in tetrahydrofuran (150 mL) wasadded triethylamine (3.03 g, 30.0 mmol), and methyl chloroformate (2.84g, 30.0 mmol) and stirred at room temperature for 1 h. To this mixturewas added diethylamine (4.4 g, 60.0 mmol) and the mixture stirred for 16h. The reaction mixture was diluted with ethyl acetate (200 mL) andwashed with water (200 mL). The organic layer was separated, dried(MgSO₄), filtered and concentrated to give 9.1 g (81%) of a mixture ofisomers at C-6 as residue. This residue was recrystallized from ethylacetate to give 1.9 g of2-{3β-(tert-butoxycarbonylamino)-4α-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]cyclopentylidine-1,3-dithiane(isomer B at C-6).

To the above solid (1.88 g, 4.0 mmol) was added methanolic hydrochloricacid (100 mL, 0.5 N) and stirred for 16 h at 50° C. The mixture was thenneutralized with methanolic sodium hydroxide and stirred for 0.5 h atroom temperature. The mixture was concentrated and the residue passedthrough a column of silica gel (100 g) using chloroform (90): methanol(9): ammonium hydroxide (1) mixture as an eluent to give 0.6 g (50%) ofmethyl3β-amino-4α-[(diethylaminocarbonyl)(methylcarbonylamino)-methyl]cyclopentancarboxylate(isomer B at C-6) as an off-white solid, mp 95° C.

Analysis: Calculated for C₁₅H₂₇N₃O₄: C, 57.49; H, 8.68; N, 13.41 Found:C, 57.38; H, 8.63; N, 13.33

To a mixture of the above amine (0.7 g, 2.23 mmol) in dimethylformamide(13 mL) were added triethylamine (0.81 g, 8.01 mmol), S-methylN,N′-bis-tert-butoxycarbonylisothiourea (714 mg, 2.46 mmol) and mercurychloride (665 mg, 2.46 mmol) and the mixture stirred at room temperaturefor 16 h. The reaction mixture was diluted with ethyl acetate (100 mL),filtered through Celite and the filtrate washed with water (2×100 mL)and brine (1×100 mL). The organic layer was dried (MgSO₄), filtered andthe filtrate concentrated. The residue was passed through a column ofsilica gel (100 g) using ethyl acetate as an eluent. The desiredfractions were combined and concentrated to give 0.7 g (56%) of amixture of isomers. The mixture was recrystallized from ether-hexanethrice to give 0.16 g (13%) of methylt-3β-{[(tert-butoxylcarbonylamino)(tert-tritoxycarbonylimino)methyl]amino}-c-4-[(diethylamino-carbonyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylate(isomer A at C-6 and C-1) as a white solid, mp 140° C.

Analysis: Calculated for C₂₆H₄₅N₅O₈: C, 56.20; H, 8.16; N, 12.60 Found:C, 55.50; H, 8.16; N, 12.48

A mixture of the above ester (0.14 g, 0.25 mmol) in tetrahydrofuran (5mL) was stirred with sodium hydroxide (1 N, 1.5 mL) at room temperaturefor 4 h. The mixture was concentrated, the residue dissolved in water (2mL), filtered through a plug of cotton and the filtrate acidified withacetic acid. The precipitate obtained was collected by filtration,washed with water and dried to give 0.11 g (81%) of the correspondingacid.

A mixture of the above acid (0.08 g, 0.15 mmol) in dichloromethane (5mL) was stirred with trifluoroacetic acid (1.0 mL) for 16 h at roomtemperature. The reaction mixture was concentrated and the residue waswashed with ether (2×20 mL). The residue was dissolved in methanol andether added. The mixture was let stand in the refrigerator for 24 h. Thesolvent was decanted and the residue washed two times with ether anddried to give 0.06 g of the trifluoroacetic acid salt of the titlecompound as a white powder, mp >110° C. (dec).

Analysis: Calculated for C₁₅H₂₇N₅O₄.CF₃COOH: C, 44.83; H, 6.20; N, 15.38Found: C, 44.71; H, 6.37; N, 14.77

Example 31

3β-{[(Amino)(imino)methyl]amino}-4α-{[(ethyl)(propyl)aminocarbonyl](methyl-carbonylamino)methyl}cyclopentancarboxylicacid (isomer A at C-6)

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentyl-idene}-1,3-dithiane(from Example 7) (0.5 g, 0.9 mmol) in tetrahydrofuran (15 mL) was addedtriethylamine (0.12 g, 1.15 mmol), and methyl chloroformate (0.11 g,1.15 mmol) and stirred at room temperature for 1 h. To this mixture wasadded ethylpropylamine (0.32 g, 3.6 mmol) and stirred for 3 h at roomtemperature. The reaction mixture was diluted with ethyl acetate (70 mL)and washed with water (75 mL) and brine (75 mL). The organic layer wasseparated, dried (MgSO₄), filtered and concentrated. The residue waspassed through a column of silica gel (50 g) using ethyl acetate:hexane(1:1) mixture as an eluent to give 0.17 g (30%) of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethylpropylaminocarbonyl)(methylcarbonyl-amino)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer A at C-6) as a white solid, mp 115-116° C.

Analysis: Calculated for C₂₉H₄₉N₅O₆: C, 55.48; H, 7.87; N, 11.15 Found:C, 55.60; H, 7.84; N, 11.23

A 0.24 g (43%) sample of isomer B at C-6 was also isolated as a whitesolid, mp 122-123° C.

Analysis: Calculated for C₂₉H₄₉N₅O₆: C, 55.48; H, 7.87; N, 11.15 Found:C, 55.57; H, 7.89; N, 11.21

A mixture of the above isomer A (0.14 g, 0.226 mmol) and hydrochloricacid in methanol (0.75 N, 6.0 mL) was stirred at room temperature for 24h. The mixture was then neutralized with 1 N sodium hydroxide and 2drops of additional 1 N sodium hydroxide added and mixture stirred for 2h. After neutralization with 1 N hydrochloric acid, the mixture wasconcentrated, salts were removed by filtration and the filtrateconcentrated. The residue was passed through a column of silica gel (20g) using ethyl acetate: hexane (3:1) as an eluent to give 0.05 g (39%)of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[(ethylpropyl-aminocarbonyl)(methylcarbonylamino)methyl]-1-cyclopentancarboxylate(isomer A at C-6).

A mixture of the above ester (0.04 g, 0.07 mmol) and sodium hydroxide (1N, 0.5 mL) was stirred at room temperature for 2 h. The reaction mixturewas diluted with water (2 mL) and filtered through a cotton plug. Thefiltrate was neutralized with acetic acid. The precipitate which formedwas collected by filtration, washed with water and dried to give 0.03 g(77%) of3β-{[(tert-butoxycarbonylamino)(tert-butoxy-carbonylimino)methyl]amino}-4α-[(ethylpropylaminocarbonyl)(methylcarbonylamino)-methyl]cyclopentancarboxylicacid (isomer A at C-6).

A mixture of the above acid (0.012 g, 0.02 mmol) in dichloromethane (2mL) was stirred with trifluoroacetic acid (0.2 mL) for 24 h at roomtemperature. The reaction mixture was concentrated and evaporated twicewith dichloromethane to give the title compound as residue [MS (ES+):356.4].

Example 32

3β-{[(Amino)(imino)methyl]amino}-4α-{[(ethyl)(propyl)aminocarbonyl](methyl-carbonylamino)methyl}cyclopentancarboxylicacid (isomer B at C-6)

A mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-4α-[(ethylpropylaminocarbonyl)(methylcarbonylamino)methyl]-1-cyclopentylidene}-1,3-dithiane(isomer B at C-6) (from Example 31) (0.18 g, 0.288 mmol) andhydrochloric acid in methanol (0.75 N, 6.0 mL) was stirred at roomtemperature for 24 h. The mixture was then neutralized with 1 N sodiumhydroxide and 2 drops of additional 1 N sodium hydroxide added and themixture stirred for 2 h. After neutralization with 1 N hydrochloricacid, the mixture was concentrated, salts were removed by filtration andthe filtrate concentrated. The residue was passed through a column ofsilica gel (20 g) using ethyl acetate:hexane (3:1) as an eluent to give0.06 g (36%) of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]-amino}-4α-[(ethylpropylaminocarbonyl)(methylcarbonylamino)methyl]-1-cyclopentancarboxylate(isomer B at C-6).

A mixture of the above ester (0.06 g, 0.1 mmol) and sodium hydroxide (1N, 0.5 mL) were stirred at room temperature for 2 h. The reactionmixture was diluted with water (2 mL) and filtered through a cottonplug. The filtrate was neutralized with acetic acid. The precipitatewhich formed was collected by filtration, washed with water and dried togive 0.045 g (80%) of3β-{[(tert-butoxycarbonylamino)(tert-butoxy-carbonylimino)methyl]amino}-4α-[(ethylpropylaminocarbonyl)(methylcarbonylamino)-methyl]cyclopentancarboxylicacid (isomer A at C-6).

A mixture of the above acid (0.03 g, 0.05 mmol) in dichloromethane (2mL) was stirred with trifluoroacetic acid (0.3 mL) for 24 h at roomtemperature. The reaction mixture was concentrated and evaporated twicewith dichloromethane to give the title compound as residue [MS (ES+):356.3 (100%)]

Examples 33-64

General preparation of amides through parallel synthesis resulting in amixture of isomers at C-1 and C-6.

To a mixture of2-{3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonyl-imino)methyl]amino}-4α-[(carboxy)(methylcarbonylamino)methyl]-1-cyclopentyl-idene}-1,3-dithiane(from Example 7) (0.093 g, 0.16 mmol) in tetrahydrofuran (3.0 mL) wasadded triethylamine (35 μL, 0.25 mmol), and methyl chloroformate (20 μL,0.25 mmol) and the mixture stirred at room temperature for 1 h. To thismixture was added the appropriate amine (0.8 mmol) and stirred for 16 h.The reaction mixture was diluted with ethyl acetate (20 mL) and washedwith water (20 mL). The organic layer was separated, dried (MgSO₄),filtered and concentrated.

To this residue was added methanolic hydrochloric acid (4.5 mL, 0.75 N),stirred for 20 h at room temperature, made basic with sodium hydroxideand stirred for 4 h at room temperature. The mixture was againneutralized with HCl, concentrated to dryness and stirred withdichloromethane (5 mL) and trifluoroacetic acid (1 mL) for 4 h. Themixture was then concentrated to dryness and the residue characterizedby mass spectrum analysis. By this method the following amides wereisolated:

Example R₁₀ R₁₀ MS (ES+) 33 CH₃ (CH₂)₂Ph 404.4 34 CH₃ (CH₂)₃CH₃ 356.4 35C₂H₅ CH₂Ph 404.4 36 CH₃ (CH₂)₅CH₃ 384.4 37 pyrrolidino 340.5 38(CH₂)₂CH₃ CH₂-cyclopropyl 382.4 39 C₂H₅ (CH₂)₂OH 358.5 40 C₂H₅ (CH₂)₃CH₃370.4 41 CH₃ (CH₂)₂OH 344.2 42 azetidino 326.4 43 H CH(CH₃)(C₂H₅) 342.344 C₂H₅

368.4 45 CH₃ CH₂CH═CH₂ 340.3 46 CH₃ CH(CH₃)₂ 342.3 47 CH₃ (CH₂)₂CH₃342.3 48 C₂H₅ (CH₂)₂CH₃ 356.4 49 H CH(C₂H₅)₂ 356.3 50 C₂H₅ C₂H₅ 342.3 51H CH(CH₃)₂ 328.4 52 H CH(CH₃)(CH₂CH₂Ph) 418.5 53 H CH(CH₃)[CH₂CH(CH₃)₂]370.4 54 H CH(CH₂OH)(C₃H₇) 372.3 55 H CH(CH₃)(C₄H₉) 370.5 56 HCH(CH₃)[(CH₂)₂C(OH)(CH₃)₂] 414.6 57 H CH(CH₃)(CH₂OCH₃) 358.0 58 HCH(C₂H₅)(CH₂OCH₃) 372.0 59 H CH(CH₃)(C₃H₇) 356.0 60 H CH(C₂H₅)(C₃H₇)384.0 61 piperidine 354.0 62 3,4-didehydropiperidino 352.0 632-methylpiperidino 368.0 64 2-ethylpiperidino 382.0

Example 65

3β-{[(Amino)(imino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclo-pentancarboxylicacid

To a suspension of propyltriphenylphosphonium bromide (0.28 g, 0.73mmol) in tetrahydrofuran (10 mL) at −78° C. was added sodiumbis(trimethylsilyl)amide (1 M/tetrahydrofuran, 0.73 mL, 0.73 mmol)dropwise. After stirring for 10 min, the reaction mixture was allowed towarm to 0° C., stirred for 20 min, and cooled to −78° C. To this mixturewas added2-{3β-(tert-butoxycarbonylamino)-4α-[(formyl)(methylcarbonyl-amino)methyl]cyclopentylidene}-1,3-dithiane(0.097 g, 0.24 mmol) (from Example 10) in tetrahydrofuran (6 mL) and thereaction mixture was stirred for 1 h. Water (10 mL) was added and thelayers were separated. The aqueous layer was extracted with ether (4×10mL). The combined organic extracts were washed with brine, dried(MgSO₄), filtered through Celite, and concentrated in vacuo to give 0.16g of crude. Purification by radial PLC (SiO₂, 50-75% ethylacetate/hexanes) furnished 0.093 g (91%) of2-{3β-(tert-butoxycarbonylamino)-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclopentyl-idene}-1,3-dithianeas a white solid, mp 175-177° C.

Analysis: Calculated for C₂₁H₃₄N₂O₃S₂: C, 59.12; H, 8.03; N, 6.57 Found:C, 59.21; H, 8.04; N, 6.51

To a stirred solution of the above solid (0.64 g, 1.5 mmol) in methanol(44 mL) at room temperature was added 6 N HCl (3.8 mL, 22.8 mmol) andthe reaction mixture was stirred for 25 h. The reaction mixture wascooled to 0° C. and sodium hydroxide (1.0 g, 25 mmol) was added. Afterstirring for 50 min at room temperature, the reaction mixture wasquenched with glacial acetic acid (0.41 mL, 7.0 mmol) and concentratedin vacuo to furnish a residue. To this residue was added ethyl acetate(15 mL) and water (10 mL) and the layers were separated. The aqueouslayer was extracted with ethyl acetate (4×15 mL). The combined organicextracts were extracts were washed with brine, dried (MgSO₄), filteredthrough Celite, and concentrated in vacuo to give 0.37 g (66%) of methyl3β-(tert-butoxycarbonylamino)-4α-[1-(1-methylcarbonylamino)pent-2-enyl]-cyclopentancarbonylate.

A mixture of the above ester (0.28 g, 0.66 mmol) and trifluoroaceticacid (1.0 mL, 13.0 mmol) in dichloromethane was stirred at roomtemperature for 5.5 h. The reaction mixture was concentrated in vacuo togive 0.29 g (100%) of methyl3β-amino-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclopentancarboxylate.

To the mixture of the above amine (0.29 g, 0.66 mmol) indimethylformamide (7 mL) was added N,N′-bis-tert-butoxycarbonyl-S-methylisothiourea (0.24 g, 0.81 mmol), triethylamine (3.0 mL, 21.5 mmol), andmercuric chloride (0.22 g, 0.81 mmol). The reaction mixture was stirredat room temperature overnight. To this mixture was added ethyl acetate(20 mL) and water (15 mL) and the layers were separated. The organiclayer was washed with brine, dried (MgSO₄), filtered through Celite, andconcentrated in vacuo to provide 0.35 g of crude. Purification by radialPLC (SiO₂, 50-75% ethyl acetate/hexanes) gave 0.214 g (64%) of methyl3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclo-pentancarboxylate.

To a mixture of the above ester (0.116 g, 0.23 mmol) in tetrahydrofuran(3.5 mL) and water (2 mL) at room temperature was added 1 N NaOH (0.6mL, 0.6 mmol). The reaction mixture was stirred for 2 h and concentratedin vacuo. The concentrate was acidified with glacial acetic acid andextracted with ethyl acetate (4×10 mL). The combined organic extractswere washed with brine, dried (MgSO₄), filtered through Celite, andconcentrated in vacuo to afford 0.114 g (100%) of3β-{[(tert-butoxycarbonylamino)(tert-butoxycarbonylimino)methyl]amino}-4α-[1-(1-methyl-carbonylamino)pent-2-enyl]cyclopentancarboxylicacid.

A mixture of the above acid (0.114 g, 0.23 mmol) and trifluoroaceticacid (0.35 mL, 4.5 mmol) in dichloromethane (8 mL) was stirred at roomtemperature for 24 h. The reaction mixture was concentrated in vacuo togive crude. Trituration with ether afforded 0.064 g (59%) of the titlecompound as a tan solid, mp 62-64° C.

Analysis: Calculated for C₁₄H₂₄N₄O₃.1.5CF₃CO₂H C, 43.68; H, 5.50; N,11.99 Found: C, 43.48; H, 5.84; N, 12.03

Example 66

3β-{[(Amino)(imino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pentyl]cyclo-pentancarboxylicacid

A mixture of Example 65 (0.021 g, 0.045 mmol) and platinum oxide (0.05g) in ethanol (6 mL) was hydrogenated at 50 psi overnight. The reactionmixture was filtered through Celite and the filtrate was concentrated invacuo to give crude. Trituration with ether afforded 0.020 g (95%) ofthe title compound as a tan solid, mp 65-67° C.

Analysis: Calculated for C₁₄H₂₆N₄O₃.1.5CF₃CO₂H C, 43.50; H, 5.91; N,11.95 Found: C, 43.63; H, 6.16; N, 12.20

Biochemistry

The in vitro assay is based on the method reported by von Itzstein etal. (EP Application 92309634.6). The neuraminidase from the HlN9 strainof influenza was obtained by the method described by Laver et al.Virology 1984, 137, p. 314-323. Values for the IC₅₀ were measured via aspectrofluorometric technique which uses the fluorogenic substrate2′-(4-methylumbelliferyl)-α-D-acetylneuramic acid. This substrate iscleaved by neuraminidase to yield a fluorescent product which can bequantified. The assay mixture contains inhibitors at variousconcentrations (four to six points) and enzyme in 32.5 mM MES[(2-(N-morpholino)ethanesulfonic acid] buffer, 4 mM CaCl₂ at pH=6.5(total volume=80 μL). The reaction is started by the addition of 20 μLof the substrate to a final concentration of 75 μM. After 10 min at 37°C., 2.4 mL of 0.1M glycine/NaOH (pH=10.2) is added to 0.1 mL of thereaction mixture to terminate the reaction. A blank is run with the samesubstrate solution with no enzyme. Fluorescence is read using anAminco-Bowman fluorescence spectrophotometer (excitation: 360 nm andemission: 450 nm) and substrate blanks were subtracted from thereadings. The IC₅₀ is calculated by plotting percent inhibition versusthe inhibitor concentration, and determination of each point isperformed in duplicate.

Crystallography

Complexes between neuraminidase and inhibitor molecules were prepared bytransferring HlN9 neuraminidase crystals into 2 mL of the phosphatebuffer solution in which the inhibitor has been dissolved. Theconcentration of the inhibitor compound was adjusted to be 2 mM. Thecrystal was allowed to equilibrate in the buffer solution for about oneday and then removed from the solution and mounted in a glass capillaryfor X-ray diffraction data collection. All X-ray intensity measurementswere recorded with a Siemens X-100 multiwire area detector on a RigakuRU-300 rotating anode generator operating at 100 mA and 50 kV and acopper anode. The crystal to detector distance was 160 mm and thedetector was offset to collect 2.4 Å data. Intensity data were measuredon 0.1 oscillation frames at 240 s of exposure per frame. Each crystalyielded 600-700 frames of data before radiation damage to the crystalsprevented further data collection.

The intensity data were processed using the XENGEN package of programs.The integrated intensities were scaled and merged to produce a finaldata set containing only unique reflections. The final data sets werecomplete to 2.5 Å resolution. All refinement was carried out using theprogram XPLOR. The starting model for refinement was the 2.0 Å refinednative N9 structure. Difference Fourier maps to 2.5 Å were calculatedusing the calculated phases from the refined model. Analysis of theelectron density maps was performed on a Silicon Graphics Indigo Extreme2 computer graphics workstation using the graphics program QUANTA.Idealized models for the inhibitor molecules were manually fitted to thedifference electron density. These inhibitor models were later includedin the XPLOR refinement.

Biological Data Inhibition of H1N9 Influenza Neuraminidase Example No.IC₅₀ (μm) 3 115 5 280 6 90 7 3800 9 600 10 600 12 7.5 13 4.3 14 40 15 5016 70 17 2000 19 16 20 8 21 1.6 23 0.47 24 4.9 25 2300 26 0.041

Dosage and Formulation

The antiviral compounds of this invention can be administered astreatment for viral infections by any means that produces contact of theactive agent's site of action with the viral neuraminidase in the bodyof a human, mammal, bird, or other animal. They can be administered byan conventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

The dosage administered will, of course, vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent and its mode and route of administration; the age, health andweight of the recipient; the nature and extent of the symptoms, the kindof concurrent treatment; the frequency of treatment; and the effectdesired. A daily dosage of active ingredient can be expected to be about0.001 to 1000 milligram (mg) per kilogram (kg) of body weight, with thepreferred dose being 0.1 to about 30 mg/kg.

Dosage forms (compositions suitable for administration) contain fromabout 1 mg to about 100 mg of active ingredient per unit. In thesepharmaceutical compositions, the active ingredient will ordinarily bepresent in an amount of about 0.5-95% by weight based on the totalweight of the composition.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups, and suspensions. It can also be administeredparenterally, in sterile liquid dosage forms. The active ingredient canalso be administered intranasally (nose drops) or by inhalation. Otherdosage forms are potentially possible such as administrationtransdermally, via a patch mechanism or ointment.

Gelatin capsules contain the active ingredient and powdered carriers,such as lactose, starch, cellulose derivatives, magnesium stearate,stearic acid, and the like. Similar diluents can be used to makecompressed tablets. Both tablets and capsules can be manufactured assustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can besugar-coated or film-coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance.

In general, water, a suitable oil, saline, aqueous dextrose (glucose),and related sugar solutions and glycols such as propylene glycol orpolyethylene glycols are suitable carriers for parenteral solutions.Solutions for parenteral administration preferably contain awater-soluble salt of the active ingredient, suitable stabilizingagents, and, if necessary, buffer substances. Antioxidizing agents suchas sodium bisulfite, sodium sulfite, or ascorbic acid, either alone orcombined, are suitable stabilizing agents. Also used are citric acid andits salts and sodium EDTA. In addition, parenteral solutions can containpreservatives, such as benzalkonium chloride, methyl- or propylparaben,and chlorobutanol.

Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

Useful pharmaceutical dosage forms for administration of the compoundsaccording to the present invention can be illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 mg of powdered activeingredient, 150 mg of lactose, 50 mg of cellulose, and 6 mg of magnesiumstearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil, or olive oil is prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100 mu of the active ingredient. The capsules are washed anddried.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit was 100 mg of active ingredient, 0.2 mg ofcolloidal silicon dioxide, 5 mg of magnesium stearate, 275 mg ofmicrocrystalline cellulose, 11 mg of starch, and 98.8 mg of lactose.Appropriate coatings may be applied to increase palatability or delayabsorption.

Moreover, the compounds of the present invention can be administered inthe form of nose drops or a nasal inhaler.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

The foregoing disclosure includes all the information deemed essentialto enable those skilled in the art to practice the claimed invention.Because the cited applications may provide further useful information,these cited materials are hereby incorporated by reference in theirentirety.

What is claimed is:
 1. A compound represented by the formula

wherein X is CH₂, O or S R₁ is H, OH, NH₂, or OR₁₁; R₉ is CO₂H, SO₃H,PO₃H₂, NO₂, esters thereof, or salts thereof;

each of R₃ and R₈ individually is H, (CH₂)_(n)CO₂R₁₀, (CH₂)_(m)OR₁₀,CON(R₁₀)_(m), (CH₂)_(n)N(R₁₀)_(m), CH(R₁₀)_(m), (CH₂)_(n)(R₁₀)_(m),CH₂CH(OR₁₀)CH₂OR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂OR₁₀, CH₂OR₁₀, CH(OR₁₀)CH₂NHR₁₀,CH₂CH(OR₁₀)CH₂NHR₁₀, CH(OR₁₀)CH(OR₁₀)CH₂NHR₁₀, orNR₁₀C(═NR₁₀)N(R₁₀)_(m); provided that at least one of R₂, R₃ and R₈ isother than H; R₄ is (CH₂)_(n)OH, (CH₂)_(n)NH₂, (CH₂)_(n)C(═NH)NH₂,(CH₂)_(n)NHC(═NR₇)NH₂, (CH₂)_(n)CN or (CH₂)_(n)N₃; R₅ is H, lower alkyl,branched chain alkyl, cyclic alkyl or CF₃; R₇ is H, OH, CN, NH₂ or NO₂;R₁₀ is H, lower alkyl, lower alkylene, branched alkyl, cyclic alkyl,substituted cyclic alkyl, (CH₂)_(n) aromatic, (CH₂)_(n)-substitutedaromatic, and when m is 2 both R₁₀ groups can also be interconnected toform an N-heterocyclic ring; R₁₁ is lower alkyl, branched alkyl, or(CH₂)_(m) aromatic; m is 1 or 2; n is 0-4; and further provided thatwhen x is O or S, R₃ and R₈ is other than CH(OR₁₀)CH(OR₁₀)CH₂OR₁₀; andpharmaceutically acceptable salts thereof.
 2. The compound of claim 1,wherein said lower alkyl group of R₅, R₁₀ and R₁₁ contains 1 to about 8carbon atoms; and said lower alkylene group contains 2 to about 8 carbonatoms.
 3. The compound of claim 1, wherein said lower alkyl group of R₅,R₁₀ and R₁₁ contains 1 to about 3 carbon atoms; and said lower alkylenegroup contains 2 to 3 carbon atoms.
 4. The compound of claim 1, whereinsaid alkyl group of R₅, R₁₀ and R₁₁ is selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, t-butyl, cyclopentyl,and cyclohexyl, the aromatic group is selected from the group consistingof phenyl and alkyl substituted aromatic groups; the substitutedcycloalkyl group contains 3-8 carbon atoms in the ring and aresubstituted with 1 or 2 alkyl groups having 1-6 carbon atoms, hydroxygroup or both; and the alkylene group is selected from the groupconsisting of vinyl, 1-propenyl, allyl, isopropenyl, 2-methyl-2-propenyland cyclopentenyl.
 5. The compound of claim 1, wherein said salt is froman acid selected from the group consisting of hydrochloric, hydrobromic,sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic,lactic, salicyclic, succinic, toluene-p-sulphonic, tartaric, acetic,citric, methanesulphonic, formic, benzoic, malonic,naphthalene-2-sulphonic, trifluoroacetic and benzenesulphonic acid. 6.The compound of claim 1, wherein said salt is a sodium or ammonium salt.7. The compound of claim 1 beingcis-3-[(methylcarbonylamino)methyl]cyclopentanecarboxylic acid or saltthereof.
 8. The compound of claim 1 beingtrans-3-amino-c-4-(methylcarbonylamino)methyl-r-cyclopentanecarboxylicacid or salt thereof.
 9. The compound of claim 1 beingtrans-3-{[(amino)(imino)methyl]amino}-c-4-[(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid or salt thereof.
 10. The compound of claim 1 being4β-{[(amino)(imino)methyl]amino}-3α-[(2-hydroxy-1-methylcarbonylamino)ethyl]-1-cyclopentanecarboxylicacid or salt thereof.
 11. The compound of claim 1 being sodium3β-{[amino)(imino)methyl]amino}-4α-[(2-hydroxy)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylate.12. The compound of claim 1 beingtrans-3-amino-trans-1-hydroxy-cis-4[(hydroxymethyl)(methylcarbonylamino)methyl]cyclopentan-r-carboxylicacid or salt thereof.
 13. The compound of claim 1 beingtrans-3-{[(amino)(imino)methyl]amino}-trans-1-hydroxy-cis-4-[(2-hydroxymethyl)(1-methylcarbonylamino)ethyl]cyclopentan-r-carboxylicacid or salt thereof.
 14. The compound of claim 1 being3β-amino-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]cyclopentancarboxylicacid or salt thereof.
 15. The compound of claim 1 being3β-{[(amino)(imino)methyl]amino}-4α-[(1-methylcarbonylamino)(2,3,4-trihydroxy)butyl]-cyclopentancarboxylicacid or salt thereof.
 16. The compound of claim 1 beingcis-3-{[(amino)(imino)methyl]amino}-trans-1-hydroxy-trans-4-[(1-methylcarbonylamino)(2-trifluoromethyl-carbonyloxy)ethyl]cyclopentan-r-carboxylicacid or salt thereof.
 17. The compound of claim 1 beingt-3-amino-c-4-[(1-methylcarbonylamino)(2-phenylmethoxy)ethyl]-t-1-hydroxycyclopentan-r-carboxylicacid or salt thereof.
 18. The compound of claim 1 beingc-3-{[(amino(imino)methyl]amino}-t-1-hydroxy-t-4-{(methylcarbonylamino){[(methyl)-(methoxy)amino]carbonyl}methyl}cyclopentan-r-carboxylicacid or salt thereof.
 19. The compound of claim 1 beingt-3-{[(amino)(imino)methyl]amino}-c-4-[(diethylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid or salt thereof.
 20. The compound of claim 1 beingt-3-amino-c-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxy-cyclopentan-r-carboxylicacid or salt thereof.
 21. The compound of claim 1 beingt-3-{[(amino)(imino)methyl]amino}-c-4-[di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-hydroxycyclopentan-r-carboxylicacid or salt thereof.
 22. The compound of claim 1 beingc-3-{[(amino)(imino)methyl]amino}-t-4-[(di-n-propylaminocarbonyl)(methylcarbonylamino)methyl]-t-1-hydroxycyclopentan-r-carboxylicacid or salt thereof.
 23. The compound of claim 1 being3β-{[(amino)(imino)methyl]amino}-4α-[(di-n-propylaminocarbonyl)(methylcarbonylamino)-methyl]cyclopentancarboxylicacid or salt thereof.
 24. The compound of claim 1 being3β-{[(amino)(imino)methyl]amino}-4α-[(methylcarbonylamino)(3-pentylaminocarbonyl)methyl]cyclopentancarboxylicacid or salt thereof.
 25. The compound of claim 1 being3β-{[Amino)(imino)methyl]amino}-4α-[(diethylaminocarbonyl)methylcarbonylamino)-methyl]cyclopentancarboxylicacid or salt thereof.
 26. The compound of claim 1 being3β-{[(Amino)(imino)methyl]amino}-4α-{[(ethyl)(propyl)aminocarbonyl](methyl-carbonylamino)methyl}cyclopentancarboxylicacid or salt thereof.
 27. The compound of claim 1 represented by theformula:


28. The compound of claim 1 being3β-{[(Amino)(imino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pent-2-enyl]cyclo-pentancarboxylicacid or salt thereof.
 29. The compound of claim 1 being3β-{[(Amino)(imino)methyl]amino}-4α-[1-(1-methylcarbonylamino)pentyl]cyclopentan-carboxylicacid or salt thereof.
 30. A composition for inhibiting influenza virusneruaminidase, comprising: a pharmaceutically acceptable carrier and anamount effective for inhibiting influenza virus neruaminidase of acompound according to claim
 1. 31. A method for inhibiting influenzavirus neruaminidase, comprising the step of: administering to a patientin need thereof a composition comprising a pharmaceutically acceptablecarrier and an amount effective for inhibiting influenza virusneruaminidase of a compound according to claim 1.